Markers for prostate cancer

ABSTRACT

This invention provides a method for determining the aggressiveness of a prostate carcinoma comprising: (a) obtaining a sample of the prostate carcinoma; and (b) detecting the presence of p27 protein in the prostate carcinoma, the absence of p27 indicating that the prostate carcinoma is aggressive. This invention also provides a method for diagnosing a beign prostate hyperplasia comprising: (a) obtaining an appropriate sample of the hyperplasia; and (b) detecting the presence of the p27 RNA, a decrease of the p27 RNA indicating that the hyperplasia is beign. This invention provides various uses of p27 in prostate cancer. Finally, this invention also provides different marker for prostate cancer.

[0001] This application is a Continuation-In-Part application ofInternational Application No. PCT/US98/25483, filed Dec. 1, 1998, whichclaims the benefit of U.S. Provisional Application No. 60/067,190, filedDec. 1, 1997, the content of which are incorporated into thisapplication by reference.

[0002] This invention was made in part with support under United StatesGovernment NIH Grant CA-DK-47650. Accordingly, the United StatesGovernment has certain rights in the invention.

[0003] Throughout this application, various references are referred towithin parentheses. Disclosures of these publications in theirentireties are hereby incorporated by reference into this application tomore fully describe the state of the art to which this inventionpertains. Full bibliographic citation for these references may be foundat the end the specification, preceding the claims.

BACKGROUND OF THE INVENTION

[0004] It has been postulated that the loss of function of a new familyof negative cell cycle regulators, which act as cyclin-dependent kinaseinhibitors and have beeen termed CKI, might lead to tumor development.CKIs fall into two families, Kip and Ink, on the basis of sequencehomology. p27^(Kip1) is implicated in G1 phase arrest by associatingwith multiple G1 cyclin-dependent kinases, abrogating their activity.However, no tumor-specific p27^(KiP1) genomic mutations have been foundin a large group of primary human cancers studied. More recently, it hasbeen reported that proteasome-mediated degradation of p27 protein occursduring the cell cycle and that this degradation is increased in a subsetof breast and colon carcinomas of poor prognosis. Purpose: The presentstudy was undertaken in order to assess for potential alterations of p27expression in benign prostatic hyperplasia (BPH) and in a wellcharacterized cohort of patients with prostatic cancer.

[0005] Inactivation of the p53 and RB tumor suppressor genes has beenimplicated in the development and progression of a number of differentcancers (1,2). It has also been postulated that the loss of function ofa new family of negative cell cycle regulators, which act ascyclin-dependent kinase inhibitors and have been termed CKI, might alsolead to tumor development (3). CKIs fall into two families, Kip and Ink,on the basis of sequence homology (4). Kip family members include p21(also known as WAF1, Cip1, or Sdi1) (5-7), p27^(KiP1) (8-10) andp57^(Kip2) (11,12). The Ink group includes four members:p16^(INK4A/MTS1/CDKN2) (13), p15^(INK4B/MTS2) (14) p18^(INK4C) (15), andp19^(INK4D) (16). p27 is a negative regulator implicated in G1 phasearrest by TGFβ, cell-cell contact, agents that elevate cyclic AMP, andthe growth inhibitory drug rapamycin (17-21). p27 associates withmultiple G1 cyclin-dependent kinases in non-proliferating cells,abrogating their activity (4, 8-10).

[0006] To assess its role as a potential tumor suppressor, thep27^(Kip1) gene was mapped to 12p12-12p13.1 and no tumor-specificgenomic mutations in a large group of primary human cancers wereobserved (22-24). More recently, it has been reported thatproteasome-mediated degradation of p27 occurs during the cell cycle andthat this degradation is increased in a subset of breast and coloncarcinomas of poor prognosis (25-28). The present study was undertakenin order to assess for potential alterations of p27 expression in BPHand in a well characterized cohort of patients with primary andmetastatic prostatic cancer.

[0007] 74 prostate carcinomas from primary and metastatic sites,representing different hormone sensitivities were analyzed. Normalprostatic tissues and cases of benign prostatic hyperplasia were alsostudied. In order to evaluate prostatic tissue of p27 null mice, eight 7month old and six greater than 12 month old littermate pairs ofwild-type and p27 knockout animals were used. Levels of expression andmicroanatomical localization of p27 protein and RNA transcripts weredetermined by immunohistochemistry and in situ hybridization withspecific antibodies and probes, respectively. Comparative analysesbetween immunohistochemistry, immunoblotting and immunodepletion assayswere also conducted in a subset of cases. Association betweenalterations in p27 expression and clinicopathological variables wereevaluated using the two-tailed Fisher's exact test. Disease relapse-freesurvivals were evaluated using the Kaplan-Meier method and the Logranktest. Distinct anomalies in the expression of p27 in benign andmalignant human prostate tissues are reported. The normal human prostateshows abundant amounts of p27 and high levels of p27 messenger in bothepithelial and stroma cells. However, p27 protein and transcripts arealmost undetectable in epithelial and stroma cells of BPH lesions. It isalso reported that p27-null mice develop hypercellular prostatic glandswhich histologically resemble human BPH. Based on these findings wepostulate that the loss of p27 expression in human prostate may becausally linked to BPH. Prostatic carcinomas can be categorized into twogroups: those that contain detectable p27 protein and those that do not.In contrast to BPH, however, both groups of prostatic carcinomas containabundant p27 transcripts. Moreover, primary prostatic carcinomasdisplaying the p27-negative phenotype appear to be biologically moreaggressive, based on their association with time to prostate specificantigen (PSA) failure following radical prostatectomy. These resultssupport the postulate that BPH is not a premalignant lesion in thepathway of prostate cancer development. Data also suggest that prostaticcarcinoma develops along two different pathways, one involving the lossof p27 and the other using other processes that circumvent the growthsuppressive effects of p27.

SUMMARY OF THE INVENTION

[0008] This invention provides a method for determining theaggressiveness of a prostate carcinoma comprising: (a) obtaining asample of the prostate carcinoma; and (b) detecting the presence of p27protein in the prostate carcinoma, the absence of p27 indicating thatthe prostate carcinoma is aggressive.

[0009] This invention also provides a method for diagnosing a beignprostate hyperplasia comprising: (a) obtaining an appropriate sample ofthe hyperplasia; and (b) detecting the presence of the p27 RNA, adecrease of the p27 RNA indicating that the hyperplasia is beign. In anembodiment, the above method further detects the protein expression ofp27 wherein this additional step may be performed before or after thedetection of the presence of the p27 RNA.

[0010] This invention provides a method for predicting the life-span ofpatient with prostate carcinoma comprising: (a) obtaining a sample ofthe prostate carcinoma; and (b) detecting the presence of p27 protein inthe prostate carcinoma, the presence of the p27 protein indicating thatthe patient can live longer than the patient who are undetectable p27protein.

[0011] This invention also provides a method for increasing thelife-span of patient with prostate carcinoma comprising inducing theexpression of p27 protein in the prostate carcinoma.

[0012] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing a nucleic acidmolecule having sequence encoding a p27 protein into the carcinoma cellunder conditions permitting expression of said gene so as to prolong thelife-span of the patient with said prostate carcinoma. In an embodiment,the nucleic acid molecule comprises a vector. The vector includes, butis not limited to, an adenovirus vector, adeno-associated virus vector,Epstein-Barr virus vector, Herpes virus vector, attenuated HIV virus,retrovirus vector and vaccinia virus vector.

[0013] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing an effective amountof p27 protein into the carcinoma cell so as to thereby prolong thelife-span of the patient with said prostate carcinoma.

[0014] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing an effective amountof a substance capable of stabilizing the p27 protein into the carcinomacell so as to thereby prolong the life-span of the patient with saidprostate carcinoma.

[0015] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount of anucleic acid molecule having sequence encoding a p27 protein and asuitable carrier.

[0016] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount ofthe p27 protein and a suitable carrier.

[0017] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount asubstance capable of stabilizing the p27 protein and a suitable carrier.

BRIEF DESCRIPTION OF THE FIGURES Figures for the First Series ofExperiments

[0018]FIG. 1

[0019] Histological analysis, immunohistochemistry, and in situhybridization of human primary and metastatic prostatic carcinomas.

[0020] (A-C) Photomicrographs of primary prostatic carcinomas processedas follows: (A) Immunohistochemical staining against p27 of a prostaticintra-epithelial neoplastic (PIN) lesion; note the intense positiveimmunoreactivities observed in the nuclei of the tumor cells growinginto the lumen. (B) Immunohistochemical staining against p27 of anotherPIN lesion showing dysplastic changes; note the intense positiveimmunostaining in the nuclei of normal epithelial cell and thelow-to-undetectable staining of the tumor cells dissecting the gland andgrowing into the lumen. (C) Undetectable levels of p27 protein in aninvasive primary prostatic carcinoma; note the staining of a normalgland trapped into the tumor.

[0021] (D-F) Photomicrographs of metastatic prostate carcinomasprocessed as follows: (D) Immunohistochemical staining against p27 of ametastatic prostate carcinoma to lymph node; note the intense nuclearstaining of both tumor cells and lymphocytes (cells in the germinalcenter display low p27 levels). (E) Immunohistochemical staining againstp27 of another metastatic prostate carcinoma to lymph node; note theintense positive immunostaining in the nuclei of lymphocytes and theundetectable levels of p27 staining on the tumor cells. (F)Immunohistochemical staining against p27 of a metastatic prostatecarcinoma to bone; note the positive immunoreactivities in the nuclei ofosteoblasts and the lack of staining of tumor cells.

[0022] (F and G) Photomicrographs of a primary invasive prostaticcarcinoma processed as follows: (F) Low-to undetectableimmunohistochemical staining against p27 in the tumor cells; note thestaining of a normal gland trapped into the tumor. (G) In situhybridization on a consecutive section from the case illustrated inpanel (F) showing high mRNA levels of p27^(KiP1) even in p27-negativetumor cells utilizing the anti-sense probe to p27^(KiP1). Originalmagnification (A) trough (F) 400×.

[0023] FIG. 2

[0024] In certain prostatic carcinomas p27 protein is a functionalcyclin-dependent kinase inhibitor. (A) Immunohistochemical stainingcorrelates with the presence of p27 by immunoblotting. Tumors #1 and #2were negative and tumor #3 positive for p27 protein expression,paralleling their IHC patterns. (B) Immunodepletion of p27 extracts.Extracts obtained from tumors #2 and #3 were subjected to sequentialdepletion with antibodies specific to p27 or a non-specific rabbitanti-mouse (RaM). Following depletion, the proteins in the supernatantswere resolved and the presence of p27 determined by immunoblotting. (C)Depletion of p27 depletes heat stable cyclin-dependent kinase inhibitoryactivity. The supernatant shown in panel B was boiled and followingclarification the soluble fraction was incubated with different amountsof recombinant cyclin E/CDK2 kinase and the degree of inhibition ofcylin E/CDK2 activity on histone H1 substrate was measured. The amountof each kinase used is shown in the panel and the bars arerepresentative activities on an arbitrary scale. Depletion with eitherRaM or p27 specific antibodies did not affect the inhibitory activity ofthe p27 negative tumor; however, depletion of p27 from the positivetumor extract completely ablated the heat stable inhibitor activity.

[0025]FIG. 3

[0026] Recurrence-free proportion analysis of patients with primaryprostate carcinoma (n=42) as assessed by time to detectable PSA.Patients who had PSA relapse were classified as failures, and patientswith PSA relapse, or those who were still alive or died from otherdisease or lost to follow-up during the study period, were coded ascensored. Time to relapse was defined as the time from date of surgeryto the endpoint (relapse or censoring). Disease relapse-free survivalswere evaluated using the Kaplan-Meier method and the Logrank test. Atrend was observed between a p27 negative phenotype and early relapse(p=0.08).

[0027]FIG. 4

[0028] Histological analysis, immunohistochemistry, and in situhybridization of human normal prostate and benign prostatic hyperplasia.

[0029] (A-C) Photomicrographs of consecutive sections of normal prostatetissue processed as follows: (A) Immunohistochemical staining againstp27; intense positive immunoreactivities are observed in the nuclei ofepithelial cells in the luminal side of the acinus, with decreasedreactivities in the nuclei of basal and stroma cells. (B) In situhybridization showing high mRNA levels of p27^(KiP1) in both epithelialand stroma cells utilizing the anti-sense probe. (C) In situhybridization utilizing the sense probe to p27^(KiP1) showing lack ofsignals in both epithelial and stroma cells.

[0030] (D-F) Photomicrographs of consecutive tissue sections of a benignprostatic hyperplastic nodule processed as follows: (D)Immunohistochemical staining against p27; note the lack or almostundetectable levels of immunoreactivity observed in the nuclei of bothepithelial and stroma cells in the luminal side of the acinus, withdecreased reactivities in the nuclei of basal and stroma cells. (E) Insitu hybridization showing low-to-undetectable p27^(KiP1) transcriptsalso in both epithelial and stroma cells utilizing the anti-sense probe;note the strong signal of the cellular inflammatory infiltrates thatserve as an internal positive control. (F) In situ hybridizationutilizing the sense probe to p27^(KiP1) showing lack of signals inepithelial and stroma cells, as well as cellular inflammatory elements.Original magnifications: (A), (B) and (C) 1000×; (D), (E) and (F) 400×.

[0031]FIG. 5

[0032] Histopathological analysis of the prostatic tissues of 12 monthold p27+/+ (A) and p27−/− (B-D) mice. Photomicrographs of tissuesections of normal prostate samples processed as follows: (A)Hematoxylin and eosin staining of a prostate gland of a p27+/+ mouseshowing well defined acini of epithelial cells surrounded by a stromacontaining few fibroblasts and poor in supportive connective tissuecomponents. (B) Hematoxylin and eosin staining of a prostate gland of ap27−/− mouse showing multiple and complex glands and hypercellular aciniof epithelial cells surrounded by fibromuscular stroma cells in aconnective tissue displaying abundant supportive components. (C and D)Hematoxylin and eosin stainings of a prostate gland of a p27−/− mouse,high power details, illustrating the complexity of the glands andabundant fibromuscular stroma elements (C), as well as thehypercellularity of the acini (D). Original magnifications: (A) and (B)200×; (C) and (D) 400×.

Figures for the Second Series of Experiments

[0033]FIG. 6. Photomicrographs of selected primary prostate carcinomacases analyzed by immunohistochemistry utilizing mouse monoclonalantibodies PAB1801 (anti-p53, A), 2A10 (anti-mdm2, B), Ab-1 (anti-p21,C), and MIBI (anti-Ki67, D). A, p53 nuclear overexpression in tumorcells. Note the positive nuclear staining of tumor cells at a perineuralinvasion site (arrow). B, mdm2 nuclear overexpression in tumor cells.C,p21 nuclear overexpression in tumor cells. D, high Ki67 proliferativeindex. Note the intense Ki67 nuclear staining of tumor cells at aperineural invasion site (arrow).

[0034]FIG. 7. Progression-free and survival curves for patients withprimary prostate cancer. The Kaplan-Meier method was used to estimateoverall disease free survival. The log-rank analysis was used to comparethe different curves. A, progression was significantly reduced inpatients with tumors displaying a p53-postive phenotype (P<0.01). B,progression was not related to mdm2 status. C, progression wassignificantly reduced in patients with tumors displaying a p21 positivephenotype (P=0.0165).

[0035]FIG. 8. Diagrammatic representation of the p53-pathway (A), andalterations that may develop during tumor progression in prostate cancer(B). (A) p53 regulates the expression of several genes involved in cellcycle arrest (ie, p21) and apoptosis (ie, bax). p21 binds toheterodimeric protein kinases formed by cyclins and cyclin-dependentkinases (Cdk's), blocking phosphorylation of pRB/E2F1 complexes andabrogating S-phase entry. p53 also produces an autoregulatory feed backloop by transactivating mdm2. (B) Overexpression of mdm2 has beenobserved to occur in several tumor types, and it is considered anoncogenic event. Upon binding to mdm2, p53 products aretrasncriptionally inactivated and triggered for degradation. This willrelease the G1 arrest imposed, in part, by p21 and abolish the apoptoticsignals of the pathway. Thus, inactivation of p53 will favorproliferative activity, immortality, and development/accumulation offurther DNA damage or mutations. The increased p21 expression observedin our study could be produced via growth factor signaling, which wouldalso impact on cyclin D1 expression. The increment of p21 does notappear to be able to control the proliferative activity of tumor cells,as attested by the association of p21 positive phenotype and high Ki67proliferative index. Taken together, mdm2 overexpression will inactivatethe p53-pathway, while increased mitogenic activity will offset theRB-pathway. The mechanistic basis for this dual requirement stems, inpart, from the deactivation of a p53-dependent cell suicide program thatwould normally be brought about as a response to uncheked cellularproliferation resulting from RB-deficiency.

Figures for the Third Series of Experiments

[0036]FIG. 9A., FIG. 9B., FIG. 9C., FIG. 9D., FIG. 9E., and FIG. 9F.

Figures for the Fourth Series of Experiments

[0037] FIGS. 10A-B. Immunohistochemistry and in situ hybridization ofhuman benign prostatic hyperplasia (BPH). Consecutive sections of benignhyperplastic prostate tissue were processed as follows: A)Immunohistochemical staining of p16 is shown. Protein expression levelsare undetectable in both epithelial and stromal components. B) In situhybridization shows undetectable mRNA levels of p16 in both epithelialand stromal components when the antisense probe is used.

[0038] FIGS. 11A-D. Immunohistochemistry and in situ hybridization ofhuman primary prostatic carcinomas. Consecutive sections of primaryhuman prostate cancer tissue were processed as follows: A)Immunohistochemical staining of p16 is shown. Lack of immunoreactionnoted in the nuclei and cytoplasm of both epithelial and stromalcomponents. B) In situ hybridization reveals undetectable mRNA levels ofp16 in both epithelial and stromal components when the antisense probeis used. (C-D) Histologic analysis, immunohistochemistry, and in situhybridization of human primary prostatic carcinoma showing p16overexpression. Consecutive sections of primary human prostate cancertissue were processed as follows: C) Immunohistochemical staining of p16is shown. Note strong brown immunoreaction observed in the nuclei ofcells. Faint cytoplasmic staining is noted as well. D) In situhybridization shows high mRNA levels of p16 in epithelial cells when theantisense probe is used. A normal gland (see pointer) serves as aninternal negative control in both the immunohistochemical analysis inFIG. 11C and also the in situ hybridization analysis in FIG. 11D.

[0039]FIG. 12. Kaplan-Meier curves, using the log rank test, stratifiedby p16 groups (group A or group B) of patients with primary prostatecarcinoma (n=88) as assessed by time to detectable PSA level postprostatectomy. Time to relapse was defined as the time from the date ofsurgery to the time of PSA elevation after surgery. The median time torelapse for group A has not been reached. The median time to relapse forgroup B was 46.25 months. Patients who had PSA relapse were classifiedas having treatment failures and tumor recurrence.

DETAILED DESCRIPTION OF THE INVENTION

[0040] This invention provides a method for determining theaggressiveness of a prostate carcinoma comprising: (a) obtaining asample of the prostate carcinoma; and (b) detecting the presence of p27protein in the prostate carcinoma, the absence of p27 indicating thatthe prostate carcinoma is aggressive.

[0041] This invention also provides a method for diagnosing a beignprostate hyperplasia comprising: (a) obtaining an appropriate sample ofthe hyperplasia; and (b) detecting the presence of the p27 RNA, adecrease of the p27 RNA indicating that the hyperplasia is beign. In anembodiment, the above method further detects the protein expression ofp27 wherein this additional step may be performed before or after thedetection of the presence of the p27 RNA.

[0042] This invention provides a method for predicting the life-span ofpatient with prostate carcinoma comprising: (a) obtaining a sample ofthe prostate carcinoma; and (b) detecting the presence of p27 protein inthe prostate carcinoma, the presence of the p27 protein indicating thatthe patient can live longer than the patient who are undetectable p27protein.

[0043] This invention also provides a method for increasing thelife-span of patient with prostate carcinoma comprising inducing theexpression of p27 protein in the prostate carcinoma.

[0044] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing a nucleic acidmolecule having sequence encoding a p27 protein into the carcinoma cellunder conditions permitting expression of said gene so as to prolong thelife-span of the patient with said prostate carcinoma. In an embodiment,the nucleic acid molecule comprises a vector. The vector includes, butis not limited to, an adenovirus vector, adeno-associated virus vector,Epstein-Barr virus vector, Herpes virus vector, attenuated HIV virus,retrovirus vector and vaccinia virus vector.

[0045] Methods to introduce a nucleic acid molecule into cells have beenwell known in the art. Naked nucleic acid molecule may be introducedinto the cell by direct transformation. Alternatively, the nucleic acidmolecule may be embedded in liposomes. Accordingly, this inventionprovides the above methods wherein the nucleic acid is introduced intothe cells by naked DNA technology, adenovirus vector, adeno-associatedvirus vector, Epstein-Barr virus vector, Herpes virus vector, attenuatedHIV vector, retroviral vectors, vaccinia virus vector, liposomes,antibody-coated liposomes, mechanical or electrical means. The aboverecited methods are merely served as examples for feasible means ofintroduction of the nucleic acid into cells. Other methods known may bealso be used in this invention.

[0046] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing an effective amountof p27 protein into the carcinoma cell so as to thereby prolong thelife-span of the patient with said prostate carcinoma.

[0047] This invention provides a method for prolong life-span of patientwith prostate carcinoma which comprises introducing an effective amountof a substance capable of stabilizing the p27 protein into the carcinomacell so as to thereby prolong the life-span of the patient with saidprostate carcinoma. Such substance may be either inhibiting the proteasewhich degrade the p27 protein or it may interact with p27 in such a waythat the protein will be resistant to degradation. By administering suchsubstance into the cell, the effective amount of p27 protein will beincreased.

[0048] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount of anucleic acid molecule having sequence encoding a p27 protein and asuitable carrier.

[0049] As used herein, the term “suitable carrier” encompasses any ofthe standard carriers. The composition may be constituted into any formsuitable for the mode of administration selected. Compositions suitablefor oral administration include solid forms, such as pills, capsules,granules, tablets, and powders, and liquid forms, such as solutions,syrups, elixirs, and suspensions. Forms useful for parenteraladministration include sterile solutions, emulsions, and suspensions.

[0050] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount ofthe p27 protein and a suitable carrier.

[0051] This invention provides a composition for prolong life-span ofpatient with prostate carcinoma which comprises an effective amount asubstance capable of stabilizing the p27 protein and a suitable carrier.

[0052] This invention provides a method for determining the rate ofproliferation of a prostate cancer comprising: (a) obtaining a sample ofthe prostate cancer; and (b) detecting the presence of p21 protein inthe prostate cancer, the presence of p21 indicating that the prostatecancer will have a high proliferation rate.

[0053] This invention also provides a method for determining the rate ofproliferation of a prostate cancer comprising: (a) obtaining a sample ofthe prostate cancer; and (b) detecting the mdm2 expression in theprostate cancer, the overexpression of mdm2 indicating that the prostatecancer will have high proliferation rate.

[0054] This invention provides a method for determining whether aprostate cancer would be metastatic comprising: (a) obtaining a sampleof the prostate cancer; and (b) detecting the level of cyclin D1expression in the prostate cancer, the overexpression of cyclin D1indicating that the prostate cancer will be metastatic. In anembodiment, the prostate cancer is metastatic to bone.

[0055] This invention provides a method for determining the tumorrecurrence in prostate cancer comprising: (a) obtaining a sample of theprostate cancer; and (b) detecting the expression of thecyclin-dependent kinase inhibitor p16 in the prostate cancer, theoverexpression of p16 indicating that the prostate cancer will have hightumor recurrence.

[0056] This invention will be better understood from the ExperimentalDetails which follow. However, one skilled in the art will readilyappreciate that the specific methods and results discussed are merelyillustrative of the invention as described more fully in the claimswhich follow thereafter.

EXPERIMENTAL DETAILS Experimental Details for First Series ofExperiments MATERIALS AND METHODS

[0057] Patient Characteristics and Tissues. A cohort of 74 patients withprostatic carcinoma were evaluated. Tissues were obtained from theDepartment of Pathology, Memorial Sloan-Kettering Cancer Center, NewYork. Samples were formalin-fixed, paraffin-embedded tissue specimens.Fourty-two primary prostate adenocarcinoma specimens were evaluated, aswell as 9 metastases to lymph node and 23 metastases to bone. Normalprostatic tissue and/or areas of benign prostatic hyperplasia adjacentto tumor were observed in the majority of the primary cases studied.These tissues were also analyzed as part of the study. In addition, 10pairs of frozen normal and tumor prostate tissues were utilized forantibody titration, as well as comparative analyses betweenimmunohistochemistry, immunoblotting and immunodepletion assays (seebelow). Representative hematoxylin-eosin stained sections were examinedto evaluate the histopathological characteristics of the lesions to beanalyzed, including the ratio of normal-to-tumor content formicrodissection techniques.

[0058] In order to evaluate prostatic tissue of p27 null mice, eight 7month old and six greater than 12 month old littermate pairs ofwild-type and p27 knockout animals were used. Tissues were dissected,weighted and processed for histology by formalin fixation and paraffinembedding. Tissue sections were cutted and stained withhematoxylin-eosin for histologic analysis. All sections were utilized tocount the number of acini per gland, a process that was conductedutilizing magnifications of 200×.

[0059] Antibodies and Immunohistochemistry. The following wellcharacterized antibodies and corresponding final working dilutions wereused for the present study: monoclonal antibody p27/Kip1 (Ab-2, OncogeneScience, Boston, Mass.—0.1 ug/ml final concentration) and anti-p27affinity purified rabbit antiserum (1:500 dilution). A non-immune rabbitserum and mouse monoclonal antibody MIgS-KpI were used as negativecontrols at similar working dilutions. Deparaffinized sections weretreated with 3% H₂O₂ in order to block endogenous peroxidase activity.Sections were subsequently immersed in boiling 0.01% citric acid (pH6.0) in a microwave oven for 15 minutes to enhance antigen retrieval,allowed to cool, and incubated with 10% normal horse or normal goat serato block non-specific tissue immunoreactivities. Primary antibodies werethen incubated overnight at 4° C. Biotinylated horse anti-mouse IgGantibodies (Vector Laboratories, Burlingame, Calif.—1:500 dilution) orgoat anti-rabbit antibodies (Vector Laboratories—1:800 dilution) wereapplied for 1 hour, followed by avidin-biotin peroxidase complexes for30 minutes (Vector Laboratories—1:25 dilution). Diaminobenzidine wasused as the final chromogen and hematoxylin was used as the nuclearcounterstain. Nuclear immunoreactivities were classified as a continuumdata (undetectable levels or 0% to homogeneous staining or 100%). Tumorswere grouped into two categories defined as follows: negative (0% orundetectable staining to <20% nuclear immunoreactivity in tumor cells),and positive (neoplasms with ≧20% tumor cells with nuclear staining)(see statistical section).

[0060] Probes and In Situ Hybridization. Digoxigenin-labeled probes wereused for in situ hybridization and 1 ug of recombinant plasmid pCR™II(Invitrogen, San Diego, Calif.), containing the full length human p27gene (gift of Dr. M. Pagano, New York University School of Medicine, NY)was linearized by BamHI and XbaI to generate antisense and sensetranscripts. Riboprobes were generated with T7 and SP6 polymerase for 2hours at 37° C. in 1× transcription buffer (Boehringer Mannheim,Indianapolis, Ind.), 20 U of RNAse inhibitor, 1 mmol/L each of ATP, GTP,CTP, 6.5 mmol/L UTP and 3.35 mmol/L digoxigenin-UTP. Deparaffinizedtissue sections were rinsed in water and PBS for 10 minutes. The slideswere digested with Proteinase K (50 ug/ml) for 18 minutes at 37° C. inPBS, and post-fixed at °4 C. in a freshly prepared solution of 4%paraformaldehyde in PBS for 5 minutes. Prehybridization was done for 30minutes at 45° C. in 50% formamide and 2×SSC. The hybridization bufferconsisted of 50% deionized formamide (v/v), 10% dextran sulphate (50%stock solution), 2×SSC (20× stock solution), 1% SDS (10% stocksolution), and 0.25 mg/ml of herring sperm DNA (10 mg/ml). Hybridizationwas peformed overnight at 45° C. applying 10 pmol/L digoxigenin-labeledriboprobe in 50 ul of hybridization buffer per section under acoverslip. The coverslips were removed and the slides were washed inpre-warmed 2×SSC for 20 minutes at 60° C. twice, followed by washes inpre-warmed 0.5×SSC and 0.01×SSC at 60° C. for 20 minutes, respectively.After these washes the slides were incubated in normal sheep serumdiluted in buffer pH 7.5 and successively in the same buffer withantibody anti-digoxigenin-AP (Boehringer Mannheim, Indianapolis, Ind.)at dilution of 1:1500 for 1 hour at room temperature. The visualizationwas accomplished by nitro-blue tetrazolium5-bromo-4-chloro-3-indoylphosphate. The slides were counterstained withmethyl green and mounted.

[0061] Immunoblotting and Immunodepletion Assays. Proteins wereextracted from three OCT-embedded prostatic carcinomas and resolved onpolyacrylamide gels for immunoblotting with p27-specific antibodies.Extracts obtained from p27 positive and negative tumors were subjectedto sequential depletion with antibodies specific to p27 or anon-specific rabbit anti-mouse (RaM). Following depletion, the proteinsin the supernatants were resolved and the presence of p27 determined byimmunoblotting. Aliquots of these supernatants were briefly boiled andfollowing clarification the soluble fraction was incubated withdifferent amounts of recombinant cyclin E/CDK2 kinase and the degree ofinhibition of cylin E/CDK2 activity on histone H1 substrate wasmeasured.

[0062] Statistical Methods. The statistical analyses were conducted asfollows. For alterations of the p27, we divided patients into twogroups: p27 negative (0% or no immunohistochemical staining to <20%tumor cells displaying nuclear reactivities) or p27 positive (≧20% tumorcells with nuclear immunostaining with IHC). The data analyses wereconducted to explore the relationship between p27 alterations andclinicopathological variables such as presentation (primary, lymph nodemetastases, and bone metastases), clinical stage (B, C, D), totalGleason score (6 or less versus 7 or more), and hormonal status (naïveversus androgen-independent) in a total 74 patients. For 42 patientswith primary prostate cancer who underwent radical prostatectomy,further analysis was conducted to evaluate the relationship between p27alterations and clinical variables, including those described above andPSA relapse (yes and no). Two-tail Fisher's exact test was utilized toassess these associations and two tailed p-values were employed as asignificant level (29). The FREQ procedure in SAS was used in this study(30). In the analysis of disease relapse-free survival, patients who hadPSA relapse were classified as lost failures, and patients with PSArelapse, or those who were still alive or died from other disease or tofollow-up during the study period, were coded as censored. Diseaserelapse-free survivals were evaluated using the Kaplan-Meier method (31)and the Logrank test (32). The LIFETEST procedure in SAS was used (30).Proportional hazards analysis was used to obtain maximum likelihoodestimates of relative risks and their 95% confidence intervals (33,34).

EXPERIMENTAL RESULTS AND DISCUSSION Experimental Results and Discussionfor the First Series of Experiments

[0063] To determine whether loss of p27 expression was a common featurein prostate cancer, we analyzed 74 prostate carcinomas from primary andmetastatic sites, representing different hormone sensitivities. Includedwere 42 hormone-naïve primary tumors, some with associated prostaticintraepithelial neoplastic (PIN) lesions, and 32 metastatic carcinomasfrom lymph node tumors (n=9) and bone metastases (n=23). Thirteen ofthese metastatic lesions were from hormone-naïve cases, while theremaining 19 metastases were obtained after hormonal treatment. PINlesions displaying a cribiform or pseudopapillary pattern expressed highlevels of p27 protein (FIG. 1A) and were associated with p27-positiveinvasive prostatic carcinomas. In contrast, PIN lesions displaying aflat growth pattern had low to undetectable p27 levels (FIG. 1B) andwere associated with p27-negative invasive tumors. Of the invasiveprimary prostatic carcinomas studied, 12 of 42 (28.5%) cases had anintense nuclear immunoreactive p27 pattern in the malignant cells (datanot shown). The remaining 30 (71.5%) primary neoplasms displayed alteredpatterns of expression: 12 cases had undetectable p27 levels (FIG. 1C),while 18 cases had a heterogeneous pattern of expression (data notshown). In metastatic lesions, 7 of 32 (21.9%) showed intense p27nuclear immunostaining in most tumor cells (FIG. 1D). The remaining 25(78.1) metastatic lesions had either heterogeneous (data not shown) orundetectable nuclear expression of p27 (FIGS. 1E and 1F). Interestingly,all but one of the nine patients with hormone-independent bone lesionsdisplayed altered p27 expression. Four of these 9 cases had undetectablep27 protein expression (FIG. 1F), 4 cases had heterogeneous patterns ofp27 expression ranging from 30% to 40% tumor cells with weak positivestaining, and one case displayed 80% positive tumor cells. However, highlevels of p27^(KiP1) mRNA, as determined by in situ hybridization to ap27 cDNA probe, were found in all tumors even when the lesions displayedundetectable levels of p27 protein (FIGS. 1G and 1H).

[0064] In the group of tumors that expressed p27, we next determined ifthe p27 protein was inactivated. To accomplish this we extracted proteinfrom fresh frozen samples and measured the heat stable Cdk inhibitoryactivity, using cyclin E/CDK2 as a substrate, remaining in extractsfollowing depletion with p27-specific antibodies as described previously(17) (FIG. 2). Depletion of p27 protein was confirmed by immunoblotting.As expected, the depletion of extracts derived from p27 negative tumorsdid not affect the heat stable inhibitory activity, nor did depletion ofp27 positive tumor extract with a non-specific rabbit-anti-mouseimmunoglobulin. However, depletion of extracts derived from p27 positivetumors with the p27-specific antibody completely removed the inhibitoryactivity, indicating that p27 was functional as a Cdk inhibitor in thesesamples.

[0065] Taken together, these data suggest that prostatic carcinomasdevelop along two different pathways, one involving the loss of p27 andthe other using alternative processes that may circumvent the growthsuppressive effects of p27. In order to determine if these distinctpathways of prostate tumorigenesis correlate with clinical parameters,as reported for other tumor types (25-28), associations between p27immunostaining, stage, total Gleason score, and hormonal status of thetumor were assessed. No associations between detectable versusundetectable p27 protein, Gleason score (6 or less versus 7 or more), orhormonal status (naïve versus androgen-independent) were observed. Toassess disease aggressiveness, we evaluated the time to PSA failure, themost sensitive indicator of success or failure following radicalprostatectomy, in patients treated for localized disease. Only patientswho had an undetectable PSA level after surgery, an indication that theresection was complete, were considered. A trend toward an associationwas observed between a p27 negative phenotype and early relapse (p=0.08)(FIG. 3). This difference did not reach statistical significance due tothe limited sample size of the cohort analyzed. Supporting this conceptis the fact that in a multivariate proportional hazards analysis, aftercontrolling for stage and Gleason score, p27 status still was thestrongest factor in predicting PSA relapse (p=0.07).

[0066] These data suggest extending the characterization of p27expression to normal prostate and benign prostatic hyperplasia. In thenormal human prostate, abundant amounts of p27 protein were detected inthe ductal and acinar cells, mainly luminal elements, as well as stromacells using immunohistochemistry. Epithelial cells displayed a strongnuclear immunostaining signal (FIG. 4A). Likewise, both epithelial andstroma cells expressed abundant p27 transcripts (FIGS. 4B and 4C), asdetected by in situ hybridization. Strikingly, in 12 cases of BPH p27expression was low to undetectable in epithelial and stroma cells in thehyperplastic nodules. Immunohistochemical staining revealed low toundetectable immunoreactivities in both epithelial and fibromuscularcells in the hyperplastic nodules (FIG. 4D). This contrasts with thestrong p27 nuclear immunostaining phenotype observed in the normalprostate. Likewise, p27 mRNA transcript levels were low to undetectableon consecutive sections of BPH by in situ hybridization (FIGS. 4E and4F). In some of these BPH tissue samples we found areas of basal cellhyperplasia. These cellular elements also had low to undetectableamounts of p27 protein and transcripts (data not shown). Nevertheless,in the non-hyperplastic regions of these same BPH samples, normal ductaland acinar epithelial cells, as well as stroma elements, showed highlevels of p27 expression. These results indicate that in the developmentof BPH, p27 transcription may be down-regulated. This finding was quiteunexpected as this gene product is generally regulated atpost-transcriptional levels (35-37), although members of the nuclearhormone receptor superfamily are suggested to regulate p27^(KIP1) mRNAlevels (38).

[0067] The targeted deletion of the p27 locus in a murine model wasrecently reported (39-41). p27 deficient mice are viable and displayorganomegaly, increased body size and female infertility. Theseanomalies could not be attributed to a defect of the growthhormone/IGF-1 axis, rather, they resulted from excess proliferationprior to withdrawal of cells into a terminally differentiated state(39). No increased incidence of spontaneous tumors was observed;however, many p27-null mice developed a pituitary hyperplasiareminiscent of adenoma in the intermediate lobe. These data suggest thatp27 deficiency leads to hyperplasia in many tissues and organs. The highfrequency of benign prostatic hyperplasia (BPH) in men and thealterations on p27 expression in that condition suggested a parallel top27 deficiency. Previous reports of histopathological analyses of p27null mice did not include the prostate (39-41). We next set up todetermine the morphologic characteristics of the prostate gland in p27deficient animals. Comparing the total mean prostate weights of 7 monthold age-matched p27+/+ (n=8) and p27−/− (n=8) mice, the differences werenot significant [mean +/− SD: 80.6 mg (+/−8.6 mg) and 90.1 mg (+/−13.3mg), respectively (p=0.1)]. However, the mean acini counts of the totalgland in these groups were significantly different [mean +/− SD: 50.4(+/−8.5) and 74.9 (+/−8.9), respectively (p<0.01)]. A similarrelationship was observed in the mean total prostate weights of the old(greater than 12 months) p27+/+ (n=6) and p27−/− (n=6) mice [mean +/−SD: 114.0 mg (+/−18.5 mg) and 119.0 mg (+/−26.8 mg), respectively(p=0.7)], and the mean acini counts [mean +/− SD: 54.7 (+/−6.5) and 73.8(+/−5.3), respectively (p<0.01)]. The significant increase in the numberof acini in both young and old p27 deficient mice was associated withhistopathological differences that became more accentuated in theelderly group. The hyperplastic prostate of the older p27−/− mice showedenlarged glands, development of hypercellular acini of epithelial cells,and an increase in fibromuscular stroma cells (FIG. 5). Thesehistological changes are reminiscent of BPH in humans and support thehypothesis that the loss of p27 expression in human prostate may becausally linked to BPH.

[0068] It has been suggested that BPH and malignant prostate growthshare a common pathway because they commonly coexist and demonstrateandrogen dependency (42-44). However, this relationship remains unclearsince BPH tends to develop in the transition zone, while the majority ofcarcinomas develop in the peripheral zone (45-48). Results from thepresent study reveal that, unlike in the BPH lesions, prostaticcarcinoma cells regulate p27 expression at the post-transcriptionallevel. Taken together these data support the postulate that BPH is not apremalignant lesion in prostate cancer development.

[0069] Coordinate inactivation of the pathways involving the p53 and RBgenes appears to be an essential requirement for the genesis of mosthuman cancers. However, both p53 mutations and RB alterations arereported to be late and uncommon events in prostate tumor progression(49-52). Contrary to these results, data from this study indicate thatinactivation of p27 is a frequent and early event in some prostatecancers. It is thus our working hypothesis that p27 represents anotherpathway of tumor suppression in certain human tumors, prostate cancerbeing a paradigm in which this concept could be further tested.

[0070] In summary, data from this study suggest that p27^(KiP1) geneablation in the mouse causes a pronounced prostatic hyperplasia, andthat the loss of p27 expression in human prostate may be causally linkedto BPH. In addition, data from this study suggest that prostaticcarcinoma develops along two different pathways, one involving the lossof p27 and the other using alternative processes that circumvent thegrowth suppressive effects of p27. These phenotypes can be identified asearly as in the PIN stage. Moreover, primary prostatic carcinomasdisplaying the p27-negative phenotype appear to be biologically moreaggressive, based on their association with time to PSA failurefollowing radical prostatectomy while controlling for other variables.The consistent alteration of p27 expression observed in allandrogen-independent metastatic lesions suggests an association withtumor progression, which may be the result of the metastatic processitself. Alternatively, it may be postulated that p27 positive tumors aremore sensitive to androgen ablation, the primary treatment of metastaticdisease. Finally, two dissimilar mechanisms appear to be involved in theloss of p27 expression in BPH versus a subset of prostatic carcinomas.p27^(KiP1) mRNA levels are extensively reduced in BPH, whereas p27proteins are diminished to undetectable levels in some prostaticcarcinomas despite detectable p27 mRNA as the result of apost-transcriptional event. These results support the postulate that BPHis not a premalignant lesion in the pathway of prostate cancerdevelopment.

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SECOND SERIES OF EXPERIMENTS

[0123] To determine the potential role of p53 inactivation in prostatecancer, we studied a well characterized cohort of 86 patients treatedwith radical prostatectomy. We analyzed patterns of p53, mdm2, andp21/WAF1 expression by immunohistochemistry. Results were thencorrelated with clinicopathological parameters of poor outcome,including time to PSA relapse. In addition, data were also correlatedwith proliferative index, as assessed by Ki67 antigen detection. p53positive phenotype, defined as identification of nuclearimmunoreactivity in >20% tumor cells, was observed in 6 of 86 cases(7%). An association was observed between p53 positive phenotype anddecreased time to PSA relapse (P<0.01). mdm2 positive phenotype, definedas ≧20% tumor cells displaying nuclear immunoreactivity, was observed in28 of 86 cases (32.5%). mdm2 positive phenotype was found to beassociated with advanced stage (P=0.009). p21 positive phenotype,defined as >5% tumor cells with nuclear immunoreactivity, was observedin 28 of 86 cases (32.5%). An association was observed between p21positive phenotype and high Ki67 proliferative index (P=0.002). Patientswith p21 positive phenotype had a significant association with decreasedtime to PSA relapse (P=0.0165). In addition, a significant associationwas found between p21 positive phenotype and co-expression of mdm2(P<0.01). Fourty-three of 86 cases (50%) were found to have one or morealterations, and patients with any alteration were found to have ahigher rate of PSA relapse (P<0.01). It is our hypothesis that a pathwayof prostate cancer progression involves p53 inactivation caused by mdm2overexpression, and that p21 transactivation in this setting is due toan alternative signaling system rather than through a p53-dependentmechanism.

[0124] p53 responds to different forms of cellular stress by targetingand activating genes involved in growth arrest and cell death. A targetof p53-induce transcription is the p21/WAF1 gene, which encodes acyclin-dependent kinase inhibitor (1). In addition, levels of p53 aretightly regulated by mdm2, which binds to p53 repressing its activityand triggering its degradation. The MDM2 gene is itself under thetranscriptional control of p53, creating an autoregulatory feedback loop(2).

[0125] Alterations in the TP53 gene appear to be uncommon in prostatecancer, and their clinical significance has not been fully investigated.A recognized limitation of most studies is that they are confined to theanalysis of p53 alterations, without analyzing other critical componentsthat regulate its functions. The MDM2 gene is amplified in a variety oftumors, and mdm2 overexpression without amplification appears to be acommon mechanism of p53 inactivation in certain cancers (3,4). Lack ofdata regarding the functional status of the p53 products encountered inthe tumors analyzed represents another drawback. It has been reportedthat p21/WAF1 gene expression may serve as an indicator of p53 activity,since p21/WAF1 is under the transcriptional control of p53. However,serum or individual growth factors, such as epidermal growth factor(EGF), and fibroblast growth factor (FGF), were shown to induce p21expression in p53-deficient cells (5,6). Thus, there are at least twoseparate pathways accounting for the induction of p21, one linked toDNA-damage recognition, and the other produced by signaling mechanismscaused by certain cellular mitogens.

[0126] In the present study, we have analyzed the patterns of p53expression and those of critical components of its pathway, namely mdm2and p21, in 86 patients with prostate cancer.

[0127] The association between these markers and clinicopathologicalparameters of poor outcome, including time to PSA relapse andproliferative index, were also examined.

EXPERIMENTAL DETAILS Experimental Details for Second Series ofExperiments MATERIALS AND METHODS

[0128] Patients. A total number of 86 patients who underwent radicalprostatectomy at Memorial Sloan-Kettering Cancer Center in the periodbetween 1990 through 1991 were studied. Patient selection was based onthe availability of both adequate clinical follow up and representativearchival pathological materials for immunohistochemical analysis. Themedian age at the time of surgery was 65 years (range 46-74). Theirmedian follow up was 64.5 months (range 10-94 months). Formalin-fixed,paraffin embedded prostate tissues were obtained from our archival tumorbank. Representative hematoxylin-eosin stained sections were examined toevaluate the histopathological characteristics of each case.

[0129] Clinicopathological parameters examined include pre-treatmentPSA, pathologic stage and Gleason score, both determined based on theradical prostatectomy specimen. Time to PSA relapse was calculated fromthe day of surgery to the first detectable PSA. PSA relapse was definedas three consecutive rise in PSA at least one week apart. Only patientswho had undetectable PSA level after surgery were included in thisanalysis.

[0130] Tumors were staged pT2 (n=51) and pT3 (n=35). Twenty-ninepatients were Gleason score <7, while 18 patients were Gleason ≧7. Insix cases, due to scarcity of tumor representation in the specimen,grade was considered to be not interpretable. Thirty-three patients(38.3%) received neoadjuvant hormone treatment preoperatively, and weredefined as hormone-treated. These patients had non-evaluable Gleasonscores. Patients who did not receive neoadjuvant hormone treatment weredefined as hormone-naive.

[0131] Monoclonal Antibodies and Immunohistochemistry. The followingwell characterized mouse monoclonal antibodies and corresponding finalworking dilutions were used for the present study: anti-p53 monoclonalantibody PAB1801 (Ab-2 clone; CalBiochem/Oncogene Science, Boston,Mass.; 1:500 dilution); anti-mdm2 monoclonal antibody 2A10 (a gift fromDr. Arnold Levine, Rockefeller University, New York, N.Y.; 1:500dilution); and an anti-p21 monoclonal antibody (Ab-1 clone;CalBiochem/Oncogene Science; 1:20 dilution). An anti-Ki67 mousemonoclonal antibody (clone MIB1; Immunotech SA, France; 1:50 dilution)was used to assess proliferative index. MIgS-Kp1, a mouse monoclonalantibody of the same subclass as the primary antibodies listed above wasused as negative control.

[0132] An avidin-biotin immunoperoxidase method was utilized. Briefly,sections were subsequently immersed in boiling 0.0% citric acid (pH 6.0)for 15 minutes to enhance antigen retrieval and incubated with primaryantibodies overnight at 4° C. Biotinylated horse anti-mouse IgGantibodies were applied for 1 h (Vector Laboratories, Burlingame,Calif.; 1:500 dilution), followed by avidin-biotin peroxidase complexesfor 30 minutes (Vector Laboratories; 1:25 dilution). Diaminobenzidinewas used as the final chromogen and hematoxylin was used as the nuclearcounterstain. Nuclear immunoreactivity were classified on a continuousscale with values that ranged from undetectable levels or 0% tohomogeneous staining or 100%.

[0133] Statistical Analysis. The three markers were analyzed both aspercentage of tumor cells and as discrete variables based on a prioricut-points. The cut-point for p53 of >20% was based on our previousanalysis of p53 alterations in bladder cancer that revealed a strongassociation between p53 point mutation and p53 nuclear accumulationin >20% of tumor cells (7,8). For mdm2, the cut-point was based on whathave been published correlating mdm2 overexpression in ≧20% of tumorcells with worse clinicopathological parameters (9,10). The sameprinciple applied to the Ki67 cut-point determination (11,12). For p21the cut-point of >5% was based on our finding that normal prostateglands lack p21 expression, and the observation of p21 nuclear stainingand presence of mitotic figures indicating high proliferative activityof the tumors.

[0134] The association of percentage of tumor cells expressing themarkers with time to PSA relapse, while adjusting for other variableswith known prognostic significance, was assessed using the Coxproportional hazards model (13). In addition, Kaplan-Meier estimation(14) was performed and the log rank test (15) employed to assess theunivariate relationship between the individual markers using cut pointsand time to PSA relapse.

[0135] The associations between Gleason group and the three biomarkerswere assessed using Fisher's exact test (16). Also, associations betweenthe three markers and variables such as Ki67 proliferative index, stage,and hormone status were also assessed using the above test.

EXPERIMENTAL RESULTS Experimental Results for the Second Series ofExperiments

[0136] Table 1 summarizes the data in relation to clinicopathologicalparameters, including pre-treatment PSA, tumor stage, Gleason tumorgrade, hormone status, proliferative index, and immunophenotype profile.FIG. 7 illustrates the univariate relationships of the three markerswith time to PSA relapse with Kaplan-Meier curves estimated. TABLE 1Summary Of Data In Relation To Immunophenotype Profile P53 p21 mdm-2 (#)(%) (#) (%) (#) (%) PSA <4 0/18 0  5/18 27  3/18 16 4-10 3/28 10  7/2825  7/28 25 >10 3/40 7.5 16/40 40 18/40 45 p value .374 .382 .060 StageT < 3 3/51 5.8 14/51 27 11/51 21 T = 3 3/35 8.5 14/35 40 17/35 48 pvalue .631 .222 .009 Gleason Score <7 0/29 0  7/29 24  7/29 24 =7 2/1811 10/18 22  9/18 50 NE 4/33 2 10/33 30 11/33 33 p value .157 .074 .190Hormone Status Naive 2/53 3 18/53 33 17/53 32 Treated 4/33 12 10/33 3011/33 33 p value .139 .725 .904 Proliferation Index Ki67 Low 5/75 620/75 26 22/75 29 High 1/11 6  8/11 72  6/11 54 p value .768 .002 .096

[0137] p53 nuclear overexpression of >20% was observed in 6 of 86 cases.The distribution of p53% expression was primarily patients expressingless than 5% p53 (n=76). The other 10 patients had varying levels ofp53% expression, indicating a very low frequency of p53 alteration inthis group of patients. There is no correlation between p53 positivephenotype and pretreatment PSA, tumor stage, tumor grade, hormonestatus, or high proliferative index. Also, there is no associationbetween p53 overexpression and p21 or mdm2 overexpression. A significantassociation was observed between p53 status determined by the cut-pointand time to PSA relapse. This association is illustrated in FIG. 7.Using the log rank test to examine the overall differences between p53negative phenotype and p53 positive phenotype revealed a statisticalsignificant difference P<0.01. This indicates an obvious PSA relapsetime advantage for patients who do not overexpress p53. However, themagnitude of this difference may not be reliably estimated due to thesmall number of patients and events in the p53 positive phenotype group.

[0138] mdm2 nuclear overexpression of ≧20% tumor cells was observed in28 of 86 cases (32.5%). mdm2 positive phenotype was associated withadvanced stage (P=0.009). In addition, mdm2 overexpression was observednot to be significant with respect to a decreased time to PSA relapse(FIG. 7). A trend was observed between mdm2 overexpression and higherpretreatment PSA (P=0.06).

[0139] p21 nuclear overexpression of >5% tumor cells was observed in 28of 86 patients (32.5%). Patients with p21 positive phenotype wereobserved to have a significant association with high Ki67 proliferativeindex (P=0.002). High Ki67 porileferative index was identified in 11 of86 patients (12.7%). Patients with p21 positive phenotype had asignificant association with decreased time to PSA relapse, asillustrated in FIG. 7. Also, p21 overexpression was associated with mdm2overexpression (P<0.0l). However, no association was observed betweenidentification of p21 and/or mdm2 positive phenotype and p53overexpression.

[0140] Forty-three of the total 86 patients had one or more alteredmarkers. Patients with any alteration (p53 or mdm2 or p21) were observedto have a higher rate of PSA relapse (P<0.01).

[0141] The multivariate relationship between the markers and time to PSArelapse was assessed using Cox proportional hazards model. It was ofinterest to examine the effect of the markers while adjusting forvariables with know prognostic significance. Both, p53 and p21 positivephenotypes were significant while adjusting for pre-treatment PSA andGleason group (P<0.01 for both markers). Examination of theoverexpression of at least one marker (p53, mdm2 or p21) with respect totime to PSA relapse showed that this variable was also significant(P<0.01) while adjusted for pre-treatment PSA and Gleason group. Tumorstage (<3 vs. ≧3) was not significant in either the univariate ormultivariate analyses, and was thus excluded from the model. The modelthat seemed to account for the most information included p53 and p21,along with pre-treatment PSA.

EXPERIMENTAL DISCUSSION Experimental Discussion for the Second Series ofExperiments

[0142] Reports dealing with the frequency of TP53 mutations and p53overexpression in prostate cancer have yielded conflicting results,alterations ranging from 2% to 65% of cases studied (17-21). Thisdiscrepancy might be explained by the relatively small number of casesand different disease stages analyzed in some reports, the distinctmethodologies employed, and the cutoff points used for evaluation of IHCresults. However, a general finding was the association between p53alterations and clinicopathological parameters of poor clinical outcome,such as high grade and late stage (18,19,22). In this study, we observeda relatively low frequency of p53 nuclear overexpression in patientswith localized prostate cancer, as previously reported (23,24). Todetermine the potential clinical relevance of identifying a p53 positivephenotype, we correlated phenotypic characteristics of the tumors withthe time to PSA relapse. This is considered the most sensitive indicatorof success or failure following radical prostatectomy in patientstreated for localized disease. Analysis of data revealed that p53overexpression was significantly associated with PSA relapse (P<0.01)and independent of pretreatment PSA and Gleason group. However, themagnitude of this difference may not be reliably estimated due to thesmall number of patients and events in the positive phenotype. We alsoobserved that all patients who received neoadjuvant hormone treatmentprior to surgery and had tumors that overexpressed p53 relapsed. Thisfinding could be due to the advanced stage at which patients presentedand were selected for treatment using this modality. Mechanistically, analtered p53 status in this setting could have conferred resistance tocastration-induced apoptosis, ultimately leading to disease relapse. Theassociation between p53 overexpression and hormone refractory prostatecancer has been reported in locally advanced and metastatic disease(25). However, to our knowledge, this is the first report to suggestthat this association might be an early event in the evolution ofhormone refractory disease in clinically localized prostate cancer. Inthe present study we also analyzed alterations affecting otherregulators of the p53 pathway in primary prostate cancer, including mdm2and p21. The MDM2 gene maps to 12q13 and is found overexpressed incertain tumors, due to its amplification as a component of an ampliconthat includes other relevant genes, such as CDK4. The MDM2 is undertranscriptional regulation by p53, and encodes a 90-kDa zinc fingerprotein (mdm2) which contains a p53-binding site (26). It has been shownthat mdm2 binds to p53, and acts as a negative regulator by inhibitingp53 transcriptional activity and targeting its degradation, thuscreating an autoregulatory feedback loop (27). In this study, nuclearmdm2 overexpression was found in 32.5% of cases. We observed that mdm2positive phenotype was significantly associated with advanced stage. Ithas been previously reported that MDM2 is not amplified on primaryprostate cancer, based on a study of 29 tumors analyzed by Southern blothybridization (28). The discrepancy between the rate of MDM2 geneamplification and protein overexpression has been described in Burkitt'slymphoma and breast cancer (29,30). Furthermore, it was observed in softtissue sarcomas that mdm2 overexpression, rather than its amplification,was associated with worse clinical outcome (10). Based on data from thisstudy, we can postulate that mdm2 overexpression is a frequent mechanismof p53 inactivation in prostate cancer, and in this context the MDM2gene can be classified as an oncogene in this setting.

[0143] The p21/WAF1 gene encodes a nuclear protein nember of thecyclin-dependent kinase inhibitory KIP family involved in senescence andcell quiescence (31). The p21/WAF1 gene is also transcriptionallyregulated by p53. However, p21 induction could also be accomplished by ap53-independent pathway. Serum or individual growth factors, such as EGFand FGF, were shown to induce p21 in p53-deficient cells (32). Based onthese data, it has been postulated that p21 induction could be activatedthrough two separate pathways. The rate of p21/WAF1 mutations in humancancer is very low (33). However, there is an association betweenaltered patterns of p21 expression and clinical outcome in certaintumors, such as bladder, colon, and heptocellular carcinomas (34-36).Lack of p21 expression in these studies was correlated with poorclinical outcome, an expected finding if one postulates that p21deficiency reflects p53 inactivation. As a corollary to this hypothesis,the p21 negative phenotype observed in the above referred studies wasusually associated with p53 alterations. However, in our study we foundthat p21 positive phenotype was significantly associated with highproliferative index and mdm2 overexpression, but not with p53 status.Moreover, patients with p21 positive phenotype had a significantassociation with decreased time to PSA relapse. p21 overexpression hasbeen reported to be associated with worse prognosis in other tumortypes, including, breast, esophageal carcinoma, and squamous cellcarcinomas of head and neck (37-39). Moreover, p21 overexpression wasfound to be associated with resistance to chemotherapy in acute myeloidleukemia and glioblastoma (40,41).

[0144] These data could be interpreted as follows (see FIG. 8). Apositive p21 phenotype could signify activation of p53 in response toDNA damage or cellular stress. This effect would result in G1 arrest ofthe prostate tumor cells expressing p21. We observed, on the contrary,an association between p21 positive phenotype and increasedproliferative activity. Thus, it is more plausible to postulate that thep21 overexpression observed is caused by a p53-independenttransactivation mechanism. In the setting of prostate cancer, thealternative mechanism could be due to mitogenic stimuli via growthfactor signaling. There is abundant evidence regarding the upregulationof growth factor receptor/ligand activity in prostate tumors (42-46). Anadditional aberration causing p53 inactivation would be required in thismodel to explain the lack of cell death and association withproliferative activity. It is our hypothesis that the increased mdm2expression discussed above provides this requirement, further supportingthe oncogenic role of mdm2 in prostate cancer.

[0145] Finally, the association between p21 and hiah proliferative indexmight also reflect deregulated cyclinD1/CDK4 activity. In fact, weobserved a strong association between p21 positive phenotype and cyclinD1 overexpression in this cohort of patients (Drobnjak et al, personalcommunication). Taken together, these data supports the concept that p21overexpression denotes an inefficient pRB control on S-phase entry.

[0146] Growth control in mammalian cells is accomplished largely by theaction of the RB protein, regulating exit from the G1 phase, and the p53protein, triggering growth arrest or apoptotic processes. In this groupof patients, there is enough evidence to suggest that both mechanismsare defective in prostate cancer. The high proliferative index reflectsthe inefficient pRB control. We postulate that this phenomenon isproduced by deregulated cyclinD1/CDK4 activity, which is associated witha p21 positive phenotype. The deactivation of a p53-dependent apoptosiscould be explained by the degradation of p53 induced by mdm2overexpression.

[0147] In sum, alterations affecting the p53 pathway are frequent eventsin prostate cancer. It is our hypothesis that a pathway of prostatecancer progression involves p53 inactivation caused by mdm2overexpression, and that p21 transactivation in this setting is due toan alternative signaling system rather than through a p53-dependentmechanism.

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THIRD SERIES OF EXPERIMENTS

[0194] Cyclin D1 is a key regulator of the G1 phase progression of thecell division cycle. There is an increasing evidence that deregulatedcyclin D1 expression is implicated in tumorigenesis and tumorprogression in certain neoplasms. The present study was conducted inorder to analyze the alterations affecting cyclin D1 in prostate cancer,as well as to assess its potential clinical significance. We studied 116cases of primary (n=86) and metastatic (n=30) prostate carcinomas usingimmunohistochemistry and a well characterized monoclonal antibody tocyclin D1. The results were correlated with proliferative index, asassessed by Ki67 antigen expression and with clinicopathologic variablesof poor prognosis. Cyclin D1 positive phenotype, defined asidentification of immunoreactivity in the nuclei of ≧20% tumor cells,was found in 26 of 116 (22%) cases. A significant association wasobserved between cyclin D1 positive phenotype and clinicopathologicparameters, such as advanced tumor stage (T≧3) (P=0.045), evidence ofbone metastases (P=0.001) and with elevated preoperative prostatespecific antigen measurements (PSA>10 ng/ml) (P=0.01) Ki67 proliferativeindex was considered high when ≧20% tumor cells displayed positivenuclear staining, a phenotype that was observed in 20 of 107 (19%)evaluable cases. Moreover, high Ki67 proliferative index was associatedwith cyclin D1 overexpression (P=0.01) These data support the hypothesisthat alterations of cyclin D1 may represent an oncogenic event in humanprostate cancer. Furthermore, it appears that cyclin D1 overexpressioncontributes to tumor progression in a subset of particularly aggressiveprostate carcinomas, especially those developing osseous metastases.

[0195] Prostate cancer has been reported to be a neoplastic disease of aslow growth rate. Nevertheless, it still represents the second leadingcause of cancer deaths in men in the United States. There is an obviousdiscrepancy between the clinical impression of a slowly growing neoplasmand tendency to produce an aggressive metastatic disease in individualpatients. The prognostic indicators of histologic grade, pathologicstage, DNA ploidy, and tumor cell proliferative index proved to be oflimited value in determining the biologic behavior of prostate cancer(1-3). Tumor suppressor genes, particularly p53 and RB, implicated inthe molecular genetics of many human malignancies, were reported to bealtered in a rather low frequency in prostate cancer (4-8).

[0196] Cell cycle transitions are controlled by functional heterodimerscomposed of a cyclin, acting as a regulatory subunit, andcyclin-dependent kinase (Cdk), which acts as the catalytic component(9). Multiple cyclins have been isolated and characterized, and atemporal map of their expression has been delineated. It is postulatedthat the complexes formed by cyclin D1 and Cdk4 govern G1 progression,while cyclin E-Cdk2 controls entry into S-phase and cyclin A-Cdk2affects the regulation through S-phase (10). Cyclin D1-Cdk4 complexesexert their function through the phosphorylation of the product encodedby the retinoblastoma gene, pRb, in order to overcome the cell cycleblock imposed by hypophosphorylated pRb (10). Several studies suggestthat gene amplification and overexpression of cyclin D1 and Cdk4 areoncogenic events in certain tumors, including breast cancer (11), headand neck tumors (12, 13), esophageal (14, 15) and colorectal carcinoma(16). We undertook this study in order to analyze patterns of cyclin D1expression in prostate cancer. The alterations identified werecorrelated with Ki67 proliferative index, as well as relevantclinicopathologic parameters, in an attempt to define their potentialbiologic significance in prostate cancer.

EXPERIMENTAL DETAILS Experimental Details for Third Series ofExperiments MATERIALS AND METHODS

[0197] Patients Characteristics and Tissues. A cohort of 116 patientswith prostate carcinoma were evaluated, consisting of 86 primary and 30metastatic cases (eight metastases to lymph node and 22 metastases tobone). All primary tumors (n=86) represented consecutive cases ofpatients who underwent radical prostatectomy at Memorial Sloan-KetteringCancer Center, in the period of 1990 and 1991. All metastatic cases(n=30) were selected on the basis of the availability of tissue in thetumor bank. Samples were formalin-fixed, paraffin embedded tissuespecimens, obtained from the Department of Pathologic at MemorialSloan-Kettering Cancer Center. Representative hematoxylin-eosin stainedsections of each paraffin block were examined microscopically to confirmthe presence of tumor, as well as to evaluate the pathologic grade andstage of the tumors analyzed. Thirty-three of 86 patients with primarycarcinoma received preoperatively neoadjuvant hormone therapy (hormonetreated), while the remaining 53 patients were not treated with suchprotocols and were considered hormone-naive. Hormone-naive primarytumors with sufficient tumor representation on tissue sections wereassigned histologic grade (n=47). Histologic grade was categorized intotwo groups: low grade (Gleason score <7), and high grade (Gleason score≧7). According to pathologic stage, cases were grouped into early (organconfined tumors, T₂), or advanced tumors (extending beyond prostaticcapsule, ≧T₃) The response variable time to prostate-specific antigen(PSA) relapse was defined as the time from radical prostatectomy to thetime of the first detectable (non zero) PSA measurement. Threeconsecutive increases of PSA were required to confirm PSA relapse. Onlypatients who had a nonmeasurable PSA after radical prostatectomy wereincluded in the analysis.

[0198] Monoclonal Antibodies and Immunohistochemistry. The followingwell characterized antibodies and corresponding final workingconcentrations were used for the present study: anti-cyclin D1 mousemonoclonal antibody (Ab-3, clone DCS-6, IgG1, Oncogene, Calbiochem,Cambridge, Mass.; 1 μg/ml); anti-Ki67 mouse monoclonal antibody (cloneMIB-1, IgG1, Immunotech, Marseille, France; 4 μg/ml). A nonspecificmouse IgG1 kappa monoclonal antibody was used as a negative control atsimilar working concentrations. Immunohistochemistry was performed on 5μm tissue sections using avidin-biotin-peroxidase method and antigenretrieval. Briefly, sections were immersed in boiling 0.01 M citric acid(pH 6.0) and heated in microwave oven for 15 minutes, to enhance epitopeexposure. After cooling to room temperature, slides were incubated with10% normal horse serum for 30 minutes. Subsequently, appropriatelydiluted primary antibodies were applied for overnight incubation at 4°C. Biotinylated horse anti-mouse IgG antibodies were used as secondaryreagents, applied for an incubation period of 30 minutes (VectorLaboratories, Burlingame, Calif.; 1:500 dilution), followed byavidin-biotin-peroxidase complexes incubated for 30 minutes (VectorLaboratories—1:25 dilution). Diaminobenzidine was used as the finalchromogen and hematoxylin as the nuclear counterstain.

[0199] Immunohistochemistry Evaluation. Nuclear immunoreactivities forboth cyclin D1 and Ki67 antigens, were classified into two categoriesdefined as follows: negative (<20% tumor cells displaying nuclearimmunostaining), and positive (≧20% tumor cells with nuclearimmunostaining). The appropriateness of this cutoff point was validatedgraphically by using predicted survival time and looking at specificimmunoreactivities as a continuum data in this group of patients.Ultimately, results were interpreted as defined above.

[0200] Statistical Methods. The baseline variables examined were PSA(ng/ml) at time of diagnosis (divided into three categories: <4, 4-10and >10), Tumor grade (Gleason score) (divided into two mutuallyexclusive categories: <7 or ≧7), Pathologic stage T (2 or ≧3), andpercent cyclin D1 and Ki67 expression. Statistical analyses wereconducted to assess: 1) the correlation between immunophenotypicvariables and clinicopathologic parameters such as: presentation status,tumor grade, pathologic stage, preoperative PSA, and hormonal status; 2)the correlation among immunophenotypic variables; 3) association betweenimmunophenotypes and PSA relapse free survival. The Mantel-Haenszelchi-square test was used to assess the associations among the differentvariables and results were considered significant if the P value was<0.05. The FREQ procedure in SAS was used for this study (17). Theassociations between time to PSA relapse and the immunophenotypes wereevaluated using the Log Rank test and Kaplan Meier estimates (18).

EXPERIMENTAL RESULTS Experimental Results for the Third Series ofExperiments

[0201] Table 2 summarizes immunohistochemical data in relation toclinicopathologic parameters. FIG. 9 illustrates the immunohistochemicalstaining patterns of cyclin D1 in representative cases of primary tumorsand bone metastases. TABLE 2 Clinicopathologic Parameters in Relation toCyclin D1 Immunoreactivity cyclin cylcin D1 − (<20%) D1 + (≧20%)Parameter N (%) N (&) Total P-value Total Patients 90 (77.6) 26 (22.4)116 Presentation Primary tms 76 (88.4) 10 (11.6) 86 LN metastases 7(87.5) 1 (12.5) 8 NS Primary tms 76 (88.4) 10 (11.6) 86 Bone metastases7 (31.8) 15 (68.2) 22 0.001 Ki67 proliferative index Low (<20%) 71(81.6) 16 (18.4) 87 High (≧20%) 11 (55.0) 9 (45.0) 20 0.01  Primarytumors 76 (88.4) 10 (11.6) 86 Tm. Grade (Gleason)* Low (<7) 27 (93.1) 2(6.9) 29 High (≧7) 17 (94.4) 1 (5.6) 18 NS Path. Stage Early (T₂) 48(94.1) 3 (5.9) 51 Advanced (≧T₃) 28 (80.0) 7 (20.0) 35 0.045 Hormonalstatus H. naive 49 (92.5) 4 (7.5) 53 H. treated 27 (81.8) 6 (18.2) 33 NSPretreatment PSA <4 ng/ml 17 (94.4) 1 (5.6) 18 4-10 ng/ml 28 (100.0) 0(0.0) 28 >10 ng/ml 31 (77.5) 9 (22.5) 40 0.01 

[0202] Cyclin D1 was expressed in ≧20% tumor cells in 26 of 116 (22%)evaluable cases, corresponding to 10 of 86 (12%) primary lesions and 16of 30 (53%) metastases. There was a statistically significantassociation between cyclin D1 overexpression and the presence of bonemetastases. We observed that 15 of 22 (68%) bone metastases lesionsoverexpressed cyclin D1, while only 10 of 86 (12%) primary tumorspresented with this positive phenotype (p=0.001). Cyclin D1overexpression was also associated with advanced pathologic stage inprimary tumors. We found that 7 of 35 (20%) tumors of advanced stage(extending beyond the prostatic capsule, ≧T₃) displayed cyclin D1nuclear overexpression, compared to only 3 of 51 (6%) organ confinedtumors (T₂) (P=0.045). Cyclin D1 was also detected at increasedpercentage of tumor cells in patients with high initial pretreatment PSAvalues. Nine of 68 (13%) patients with PSA ≧10 ng/ml were cyclin D1positive compared to only 1 of 18 (5%) patients with PSA <4 ng/ml(P=0.01). There was no association between cyclin D1 overexpression andtumor grade (Gleason score) or hormonal status (hormone naive vs.hormone treated). In order to assess disease progression we evaluatedthe time to PSA failure after radical prostatectomy. There was noassociation between cyclin D1 nuclear overexpression and early relapseas defined by increased PSA measurements after radical prostatectomy inthese group of patients (p=0.2).

[0203] Cyclin D1 overexpression correlated well with high Ki67proliferative index, which was scored as being high in 20 of 107 (19%)evaluable tumors. Nine of 20 (45%) tumors displaying high Ki67proliferative index also possessed cyclin D1 nuclear overexpression,while only 16 of 87 (18%) cases with low Ki67 proliferative indexoverexpressed cyclin D1 (P=0.01). Nevertheless, Ki67 proliferative indexalone was not associated with clinicopathologic parameters of pooroutcome in this cohort of patients.

EXPERIMENTAL DISCUSSION Experimental Discussion for the Third Series ofExperiments

[0204] Autopsy records show that by the age of 80, approximately 60-70%of men around the world, have histologic evidence of prostatic carcinoma(19). Although this fact indicates generally slow growing nature of thismalignancy, there are vast differences in the progression rate anddevelopment of clinically evident or metastatic disease in thesepatients during their lifetime. Prostate cancer progression tends tofollow periprostatic and perivascular penetration, invasion alongperineural spaces, pelvic lymph node metastases and particularly bonemetastases (20). Almost one fourth of newly diagnosed cases presentswith lymph node and/or osseous metastases and only one fourth of thosesurvive five years (21). We were interested in analyzing the molecularevents that might be responsible for the progression of prostate cancerfrom indolent to a life threatening, metastatic disease.

[0205] Some earlier reports on determining prostate tumor proliferation,measured by flow cytometric S-phase fraction showed a positivepredictive value of this variable and prostate cancer progression (2).Tumors that demonstrate a higher proliferation rate are more likely togrow to and beyond prostatic capsule and to produce distant metastases.Recently, the cell division cycle regulatory mechanisms and theironcogenic role have become a major focus of cancer research. There isever growing literature on cyclins and their associated kinases andtheir role in tumorigenesis (22). Particularly D-family cyclins wereimplicated in specific human tumors. Bartkova et al. (23) report on alarge group of various human malignancies, including carcinoma of thebreast, uterus, colon, melanomas and soft tissue sarcomas, highproportion of which exhibit immunoreactivity for cyclin D1. By far themost frequent chromosomal abnormality that affects cyclin D1 in majorityof tumors is DNA amplification that results in increased expression ofthe RNA transcripts and protein levels (24). In some tumor types,however, immunohistochemistry proved to be the most accurate techniquein determining deregulated expression of cyclin D1 (11, 23). In thisstudy we used immunohistochemistry to determine cyclin D1 expression inpatients with prostate carcinoma. To our knowledge this first expressionstudy on cyclin D1 in both primary tumors and bone metastases specimens.Only recently Kallakury et al (25) evaluated the expression ofp34^(cdc2) and cyclin D1 in patients with radical prostatectomy. Resultswere correlated with conventional markers of poor prognosis. The authorsshowed no association between cyclin D1 immunoreactivity andclinicopathologic parameters, such as tumor grade, pathologic stage,lymph node metastases and with disease free survival. Our data in thisstudy, on the other hand, suggest the involvement of cyclin D1 in theprogression of human prostate cancer. There was a remarkably significantdifference in the levels of cyclin D1 expression between bone metastasesand primary tumors. There was an association between cyclin D1immunoreactivity and tumors with advanced pathologic stage. Cyclin D1further correlated well with high Ki67 proliferative index. Takentogether, these results support the theory that increased levels ofcyclin D1 expression contribute to cell cycle imbalance with extremelyshortened G1 phase and possibly with reduced cell requirements forgrowth factors to proliferate (26, 27). Subclones of tumor cells withelevated cyclins expression may acquire uncontrolled growth advantageand contribute to tumor progression.

[0206] In this study we conclude that cyclin D1 may play an oncogenicrole in prostate cancer. Our data indicate that cyclin D1 is involved intumor progression, particularly in a development of bone metastases.However, in order to determine the timeframe and prognostic value ofthis marker in prostate cancer, from the early onset to the evolution ofmetastatic disease, we intend to evaluate cyclin D1 immunophenotypes inpaired samples of primary tumors and metastatic sites from the samepatients.

REFERENCES FOR THIRD SERIES OF EXPERIMENTS

[0207] 1. Gleason, D. F. Histologic grading of prostate cancer: aperspective. Hum. Pathol., 23:273-279, 1992.

[0208] 2. Visakorpi, T., Kallionemi, O-P., Paronen, I. Y. I., Isola, J.J., Heikkinen, A. I., and Koivula, T. A. Flow cytometric analysis of DNAploidy and S-phase fraction from prostatic carcinomas: implications forprognosis and response to endocrine therapy. Br. J. Cancer, 64: 578-582,1991.

[0209] 3. Bubendorf, L., Sauter, G., Moch, H., Schmid, H.-P., Gasser, T.C., Jordan, P., and Mihatsch, M. J., Ki67 labeling index: an independentpredictor of progression in prostate cancer treated by radicalprostatectomy. J. Pathol., 178:437-441, 1996.

[0210] 4. Isaacs, W. B., Carter, B. S., and Ewing, C. M. Wild-type p53suppresses growth of human prostate cancer cells containing mutant p53alleles. Cancer Res., 51:4716-4720, 1991.

[0211] 5. Bookstein, R., Rio, P., Madreperla, S. A., Hong, F., Allred,C., Grizzle, W. E., and Lee, W.-H. Promoter deletion and loss ofretinoblastoma gene expression in human prostate carcinoma. Proc. Natl.Acad. Sci. USA, 87:7762-7766, 1990.

[0212] 6. Visakorpi, T., Kallionemi, O.-P., Heikkinen, A., Koivula, T.,and Isola, J. Small subgroup of aggressive, highly proliferativeprostatic carcinomas defined by p53 accumulation. J. Natl. Cancer Inst.,84:883-887, 1992.

[0213] 7. Bookstein, R., MacGrogan, D., Hilsenbeck, S. G., Sharkey, F.,and Allred, C. D. p53 is mutated in a subset of advanced-stage prostatecancers. Cancer Res., 53:3369-3373, 1993.

[0214] 8. Navone, N. M., Troncoso, P., Pisters. L. L., Goodrow, T. L.,Palmer, J. L., Nichols, W. W., von Eschenbach, A. C., and Conti, C. J.p53 protein accumulation and gene mutation in the progression of humanprostate carcinoma. J. Natl. Cancer Inst., 85:1657-1669, 1993.

[0215] 9. Sherr, C. J. G1 phase progression: cycling on cue. Cell,79:551-555, 1994.

[0216] 10. Matsushime, H., Quelle, D. E., Shurtleff, S. A., Shibuya, M.,Sherr, C. J., and Kato, J.-Y. D-type cyclin-dependent kinase activity inmammalian cells. Mol. Cell Biol., 14:2066-2076, 1994.

[0217] 11. Gillett, C., Fantl, V., Smith, R., Fisher, C., Bartek, J.,Dickson, C., Barnes, D., and Peters, G. Amplification and overexpressionof cvclin D1 in breast cancer detected by immunohistochemical staining.Cancer Res., 54:1812-1817, 1994.

[0218] 12. Jares, P., Fenandez, P. L., Campo, E., Nadal, A., Bosch, F.,Aiza, G., Nayach, I., Traserra, J., and Cardesa, A. PRAD-1/Cyclin D1gene amplification correlates with messenger RNA overexpression andtumor progression in human laryngeal carcinomas. Cancer Res.,54:4823-4827, 1994.

[0219] 13. Michalides, R., van Veelen, N., Hart, A., Loftus, B.,Wientjens, E., and Balm, A. Overexpression of cyclin D1 correlates withrecurrence in a group of forty-seven operable squamous cell carcinomasof the head and neck. Cancer Res., 55:975-978, 1995.

[0220] 14. Jiang, W., Kahn, S. M., Tomita, N., Zhang, Y.-J., Lu, S.-H.,and Weinstein, B. I. Amplification and expression of the human cyclin Dgene in esophageal cancer. Cancer Res., 52:2980-2983, 1992.

[0221] 15. Naitoh, H., Shibata, J., Kawaguchi, A., Kodama, M., andHattori, T. Overexpression and localization of cyclin D1 mRNA andantigen in esophageal cancer. Am. J. Pathol., 146:1161-1169, 1995.

[0222] 16. Zhang, T., Nanney, L. B., Luongo, C., Lamps, L., Heppner, K.J., DuBois, R. N., and Beauchamp, R. D. Concurrent overexpression ofcyclic D1 and cyclin-dependent kinase 4 (Cdk4) in intestinal adenomasfrom multiple intestinal neoplasia (Min) mice and human familialadenomatous polyposis patients. Cancer Res., 57:169-175, 1997.

[0223] 17. SAS Institute Inc., SAS/STAT user guide, version 6 Cary N.C.SAS Institute Inc., 1990.

[0224] 18. Kaplan, E. L., and Meier, P. Nonparametric estimation fromincomplete observations. J. Am. Stat. Assoc., 53:457-481, 1958.

[0225] 19. Carter, B. H., Piantadosi, S., and Isaacs, J. T. Clinicalevidence for and implications of the multistep development of prostatecancer. J. Urol., 143:742-746, 1990.

[0226] 20. Raghavan, D., Scher, H. I., Leibel, S. A., and Lange, P.Principles and practice of genitourinary oncology. Lippincott-Ravenpublishers. Philadelphia. New York, 1997.

[0227] 21. Murphy, G. P., Mettlin, C., Menck, H., Winchester, D. P., andDavidson, A. M. The national survey of prostate cancer in the UnitedStates by the American College of Surgeons. J. Urol., 127:928-934, 1982.

[0228] 22. Hall, M., and Peters, G. Genetic alterations of cyclins,cyclic-dependent kinases, and cdk inhibitors in human cancer. Adv.Cancer Res., 68:67-108, 1996.

[0229] 23. Bartkova, J., Lukas, J., Strauss, M., and Bartek, J. CyclicD1 oncoprotein aberrantly accumulates in malignancies of diversehistogenesis. Oncogene, 10:775-778, 1995.

[0230] 24. Lammie, G. A., Fantl, V., Smith, R., Schuuring, E., Brookes,S., Michalides, R., Dickson, C., Arnold, A., and Peters, G. D11S287, aputative oncogene on chromosome 11q13, is amplified and expressed insquamous cell and mammary carcinomas and linked to BCL-1. Oncogene,6:439-444, 1991.

[0231] 25. Kallakury, B. V. S., Sheehan, C. E., Ambros, R. A., Fisher,H. A. G., Kaufman, R. P., and Ross, J. S. The prognostic significance ofp34^(cdc2) and cyclic D1 protein expression in prostate adenocarcinoma.Cancer, 80:753-763, 1997.

[0232] 26. Jiang, W., Kahn, S. M., Zhou, P., Zhang, Y. J., Cacace, A.M., Infante, A. S., Doi, S., Santella, R. M., and Weinstein, I. B.Overexpression of cyclin D1 in rat fibroblasts causes abnormalities ingrowth control, cell cycle progression and gene expression. Oncogene,8:3447-3457, 1993.

[0233] 27. Quelle, D. E., Ashmun, R. A., Shurtleff, S. A., Kato, J. Y.,Bar-Sagi, D., Roussel, M. F., and Sherr, C. J. Overexpression of mouseD-type cyclins accelerates G1 phase in rodent fibroblasts. Genes Dev.,7:1559-1571, 1993.

FOURTH SERIES OF EXPERIMENTS

[0234] The INK4A gene maps to the region 9p21, and was initiallydescribed as encoding a 148 amino acid protein termed p16. The p16protein associates exclusively with Cdk4 and Cdk6, inhibiting theircomplexation with D-type cyclins, and the consequent phosphorylation ofpRB. This contributes to cell cycle arrest. The purpose of the presentstudy was to evaluate patterns of p16 expression in a well characterizedcohort of prostatic adenocarcinomas, while exploring potentialassociations between alterations of p16 and clinicopathologicalvariables.

[0235] Normal and malignant tissues from 88 patients with prostatecarcinoma were examined. In situ hybridization and immunohistochemistryassays were used to determine the status of the INK4A exon 1αtranscripts and levels of p16 protein, respectively. Associationsbetween altered patterns of expression and clinicopathologicalvariables, including pre-treatment prostate-specific intigen (PSA)level, Gleason grade, pathologic stage, and hormonal status, wereevaluated using the Mantel-Haenszel chi-square test. Biochemical (PSA)relapse after surgery was evaluated using the Kaplan-Meier method andthe Log rank test. The levels of p16 expression and INK4A exon 1αtranscripts in normal prostate and benign hyperplastic tissues wereundetectable. However, p16 nuclear overexpression was observed in 38(43%) prostate carcinomas, while the remaining 50 (57%) cases showedundetectable p16 levels. Overexpression of p16 protein was found tocorrelate with increased INK4A exon 1α transcripts. Moreover, p16overexpression was associated with a higher pre-treatment PSA level(P=0.018), the use of neoadjuvant androgen ablation (P=0.001), and asooner time to PSA relapse after radical prostatectomy (P=0.002). Thesedata suggest that p16 overexpression is associated with tumor recurrenceand a poor clinical course in patients with prostate cancer.

[0236] The INK4A gene maps to the short arm of chromosome 9 (9p21), andwas initially described as encoding a protein of Mr 15,845, termed p16.(1,2). The p16 protein forms binary complexes exclusively with Cdk4 andCdk6, inhibiting their kinase activity and subsequent pRbphosphorylation during the G₁ phase of the cell cycle. (1,3). Additionalcomplexity results from the presence of a second INK4A product termedp19^(ARF). (4-6) The p19^(ARF) protein nas recently been shown tointeract with mdm2 and to block mdm2-induced p53 degradation andtransactivational silencing(7,8). The two products, p16 and p19^(ARF),share exons 2 and 3 of the INK4A gene, but have distinct promoters andexon 1 units, exon 1α (p16) and exon 1β (p19^(ARF)). The INK4A gene ismutated in a wide variety of tumor cell lines and certain primary tumors(2, 9-14). In addition, methylation of the 5′ CpG island of the exon 1αpromoter region is a frequent mechanism of p16 inactivation in primarytumors (15, 16).

[0237] In prostate cancer the role of INK4A has not been wellelucidated, though analyses utilizing microsatellite markers in thevicinity of the INK4A gene have revealed loss of heterozygosity in asubset of primary and metastatic prostate tumors (17). Unlike reports ofother primary tumors, INK4A inactivation, either through deletions,mutations, or through promoter methylation, appears to be an infrequentevent in prostate cancer (18-23). The present study utilizesimmunohistochemical and in situ hybridization assays to examine patternsof p16 expression in a well characterized cohort of prostate cancerpatients treated with radical retropubic prostatectomy. Associationsbetween altered p16 phenotypes and clinicopathological variables werealso studied to further define their potential implications in prostatecancer.

EXPERIMENTAL DETAILS Experimental Details for Fourth Series ofExperiments MATERIALS AND METHODS

[0238] Patient Characteristics and Tissues. A cohort of patients withprostatic adenocarcinoma undergoing radical prostatectomy at theMemorial Sloan-Kettering Cancer Center from 1990-1991 wasretrospectively evaluated. A total of 88 patients had adequate clinicalfollow-up and available pathological materials. The median age at thetime of surgery was 65 years (range 46-74 years) The median follow-uptime was 64.5 months (range 10-94 months) Formalin-fixed,paraffin-embedded prostate tissues were obtained from the Department ofPathology. Representative hematoxylin-eosin stained sections wereexamined to evaluate the histopathological characteristics of eachtissue section.

[0239] Clinicopathologic parameters examined included pre-treatment PSA,pathologic stage (24) and Gleason grade (25), both determined based onthe radical prostatectomy specimen. Hormonal status of the patients wasalso evaluated. A portion of the cohort (34 patients—39%) was treatedwith neoadjuvant androgen ablation and were defined as hormone-treated.Patients who did not receive neoadjuvant therapy were defined as hormonenaive. Additionally, biochemical relapse was examined. Relapse wasdefined as an elevation in the serum PSA level in a patient who hadpreviously demonstrated an undetectable PSA level post-prostatectomy.That is, only patients who had an undetectable PSA level after surgerywere included in the cohort, as this indicated that the surgicalresection was complete and the patient was free of disease. Patients whohad PSA relapse were classified as treatment failures with tumorrecurrence.

[0240] Immunohistochemistry. An avidin-biotin immunoperoxidase assay wasperformed on formalin-fixed, paraffin-embedded tissue sections.Deparaffinized sections were treated with 1% H₂O₂ in order to blockendogenous peroxidase activity. Sections were subsequently immersed inboiling 0.01% citric acid (pH 6.0) in a microwave oven for 15 minutes toenhance antigen retrieval, allowed to cool, and incubated with 10%normal horse serum (Organon Tecknika Corp, Westchester, Pa.), to blocknon-specific tissue immunoreactivities. A well characterized antibody top16 (Ab-1, Oncogene Research Products, Cambridge, Mass.; 2 ug/ml finalconcentration) was then incubated overnight at 4° C. Biotinylated horseanti-mouse IgG antibodies (Vector Laboratories, Inc., Burlingame,Calif.; 1:25 final dilution) were utilized as the secondary reagents.This was followed by avidin-biotin immunoperoxidase complexes (1:25,Vector Laboratories, Inc.) for 30 minutes. Diaminobenzidine was used asthe final chromogen and hematoxylin was used as the nuclearcounterstain. Immunoreactivities, assessed in tissue sections from asingle representative block in each case, were classified as a continuumof data from undetectable levels or (0%) to homogenous staining levels(100%). Data was independently obtained by two observers, with minorinter-observer variability which was resolved by review of the problemcases. Tumors were grouped into two categories defined as follows: GroupA (≦5% nuclear immunoreactivity in tumor cells) and Group B (>5% nuclearimmunoreactivity in tumor cells).

[0241] In Situ Hybridization. Primers specific for the exon 1α sequenceof the INK4A gene were utilized to create digoxigenin-labeled probes forin situ hybridization. Probes were cloned into a PCR-Script recombinantplasmid (Stratagene, La Jolla, Calif.). Plasmid DNA (1 ug) waslinearized using BamHI and XhoI. Antisense and sense riboprobes weregenerated from in vitro transcription of the linearized DNA using T7 andT3 RNA polymerases, respectively. Transcription was sustained for 2hours at 37° C. in 1× transcription buffer (Boehringer Mannheim,Indianapolis, Ind.), 20 U of RNAse inhibitor, 10 mmol/L each of ATP,GTP, CTP, 6.5 mmol/L UTP and 3.5 mmol/L digoxigenin-UTP. Deparaffinizedtissue sections were rinsed in water and PBS for 10 minutes. The slideswere digested with Proteinase K (50 ug/ml) for 18 minutes at 37° C. inPBS, and post-fixed at 4 C. in a freshly prepared solution of 4%paraformaldehyde in PBS for 5 minutes. Prehybridization was done for 30minutes at room temperature (RT) in 50% formamide and 2× sodiumchloride/sodium citrate (SSC). The hybridization buffer consisted of 50%deionized formamide (v/v), 10% dextran sulphate (50% stock solution),2×SSC (20× stock solution), 1% SDS (10% stock solution), and 0.25 mg/mlof herring sperm DNA (10 mg/ml).

[0242] Hybridization was performed overnight at 45° C. applying 10pmol/L digoxigenin-labeled riboprobe in 50 ul of hybridization bufferper section under a coverslip. The coverslips were removed and theslides were washed in pre-warmed 2×SSC for 20 minutes at 42° C. twice,followed by washes in pre-warmed 1×SSC and 0.5×SSC at 42° C. for 20minutes. After these washes the slides were incubated in normal sheepserum diluted in buffer pH 7.5 and successively in the same buffer withanti-digoxigenin-AP antibody (Boehringer Mannheim) at a dilution of1:500 for 1 hour at RT. The visualization was accomplished by nitro-bluetetrazolium 5-bromo-4-chloro-3-indoylphosphate. The slides werecounterstained with methyl green and mounted.

[0243] INK4A exon-1α transcript levels were examined in a subgroup of 21cases. Consecutive tissue sections were used to analyze p16 protein byimmunohistochemistry and INK4A exon-1α transcript levels by in situhybridization. As in the immunohistochemical analysis, tumors weregrouped into two categories defined by the absence (≦5% tumor cells withcytoplasmic staining) or presence (>5% tumor cells with cytoplasmicstaining) of transcripts.

[0244] Statistical Methods. The statistical analyses of the data fromthe 88 primary prostate cancer patients were conducted as follows. Theresponse variable, time to PSA relapse, was defined as the time fromradical prostatectomy to the time of first detectable PSA measurement.Patients who did not achieve a non-measurable PSA after radicalprostatectomy were excluded from the analysis. Patients who were stillalive at the time of analysis without relapse were censored at the dateof last follow-up. The baseline variables examined were PSA measurementat time of diagnosis, hormone status, Gleason score (hormone naïvepatients only), stage of disease, and percent p16 expression.

[0245] Associations between p16 expression and different categoricalvariables (hormone status, tumor grade, tumor stage, and pretreatmentPSA levels) were assessed by Mantel-Haenszel chi-square test Continuousvariables, such as pre-treatment PSA, not known to follow a particulardistribution were compared between two or more groups using Wilcoxonnon-parametric tests.

[0246] The Cox proportional hazards model was used to examine themultivariate relationship between PSA relapse-free time fromprostatectomy and the baseline variables listed above. The final modelwas determined using the “all subsets” procedure in SAS PHREG and theScore criterion (26). As normal and benign tissues showed little to nop16 expression, positive expression was described as >5% nuclearexpression. This cutpoint was specified a priori and used for thesubsequent statistical analysis. Immunohistochemical and in situhybridization studies were completed, analyzed, and recorded blind toclinical information. Kaplan-Meier estimates of relapse-free survivalstratified by p16 classification were evaluated. The LIFETEST procedurein SAS was used to generate the Kaplan-Meier estimates and the resultingsurvival curves (26, 27). The Log rank test was used to test thehypothesis of no survival differences between p16 positive and p16negative populations.

EXPERIMENTAL RESULTS Experimental Results for the Fourth Series ofExperiments

[0247] The normal human prostate displayed undetectable levels of p16protein and INK4A exon 1α transcripts in ductal and acinar epithelialcells. A lack of p16 immunoreactivity in these cells was observed inhormone-treated and hormone naive cases. Fibromuscular stroma cells alsoshowed undetectable exon 1α transcripts levels. A similar negativepattern of p16 expression was observed upon the examination of prostatictissue affected with benign hyperplasia (FIG. 10).

[0248] To determine the frequency and potential clinical implications ofp16 alterations in prostate cancer, we analyzed a cohort of 88 primaryprostate carcinomas. Two patterns of p16 protein expression were noted.We observed that 50 of the 88 cases (57%) had very low (≦5% nuclearimmunoreactivity; 7 cases) or undetectable (43 cases) levels of p16protein expression (Group A) (FIG. 11A). In a subgroup of these cases wealso performed in situ hybridization assays, which revealed that allcases had undetectable INK4A exon 1α transcripts (FIG. 11B). However, wenoted that 38 of the 88 cases (43%) displayed nuclear staining withanti-p16 specific antibodies (Group B) (FIG. 1C). Immunoreactivities intumor cells were further stratified into three categories: 6% to 29%nuclear staining (n=11 cases); 30% to 59% nuclear staining (n=15 cases);and 60% to 100% nuclear staining (n=12 cases). In a subset of thesepatients, we also conducted in situ hybridization assays with the INK4Aexon 1α specific probe. All cases displaying positive immunoreactivitiesalso displayed moderate to high levels of exon 1α transcripts (FIG.11D).

[0249] Table 3 summarizes the associations between p16 phenotypes andclinicopathological variables, which were assessed by Chi-squareanalyses. Immunohistochemical detection of p16 was not associated withGleason grade, described as either low (Gleason grade 4-6) or high(Gleason grade 7-10) (P=0.153). Similarly, no association was observedbetween p16 nuclear expression and pathologic stage, defined asorgan-confined (T₁, T₂) and non organ-confined (T₃, T₄, or lymph node+)(P=0.087). However, there was a strong association between p16 nuclearexpression and pre-treatment PSA levels, based on cutoff points of <4,4-10, and >10 ng/ml (P=0.018). A similar result was observed when PSAwas assessed as a continuous variable (P=0.01). In addition, we noted asignificant correlation between p16 nuclear expression and the use ofneoadjuvant androgen ablation (P=0.001). TABLE 3 Association of p16Immunoreactivity with Tumor Grade, Hormonal Status, Tumor Stage, andPreoperative PSA Levels P16 Immunoreactivity (& of patients) Number ofP- Subjects ≦5% 5% value All Subjects 88 50 (57%) 38 (43%) GleasonGrade* 82 <7   30 (37) 24 (80)  6 (20) p = 0.153 ≧7   18 (22) 11 (61)  7(39) unable to evaluate 34 (41) Hormonal Status 88 hormone naive 54 (61)40 (74) 14 (26) p = 0.001 hormone-treated 34 (39) 10 (29) 24 (71)Pathologic Stage 88 T₂ 53 (60) 34 (64) 19 (36) p = 0.087 ≧T₃ 35 (40) 16(46) 19 (54) Pretreatment 88 PSA (ng/ml) <4.0 18 (20) 14 (78)  4 (22)4-10 29 (33) 19 (66) 10 (34) p = 0.018 >10  41 (47) 17 (41) 24 (59)

[0250] A strong association was also found between p16 nuclearoverexpression and tumor recurrence, as defined by biochemical (PSA)relapse. Increasing p16 expression correlated with an increased relativehazard of relapse, suggesting a continuous relationship of the data.Overall, tumor recurrence was observed in 34 of 88 cases (39%). Thirteenof 50 cases (26%) with undetectable-to-low p16 expression (Group A)developed tumor recurrence. However, tumor recurrence was observed in 21of 38 cases (55%) with p16 nuclear overexpression (Group B) (P=0.002)(FIG. 12). Nevertheless, in a multivariate analysis adjusted for tumorgrade, pre-treatment PSA, and pathologic stage, overexpression of p16did not contribute prognostic information over pre-treatment PSA, thestrongest independent predictor of tumor recurrence.

Experimental Discussion for the Fourth Series of Experiments

[0251] Normal prostate tissues display undetectable levels of p16protein and INK4A exon 1α transcripts. It has been reported that p16expression is low to undetectable in most normal human tissues analyzed(15, 28, 29). In support of these observations, there are relatively lowand near-constant levels of p16 protein and mRNA throughout the cellcycle of normal lymphocytes in culture (30). The lack of p16 expressionin hyperplastic glands parallels that of normal prostatic tissue. Basedon these data, it is our hypothesis that these negative p16 phenotypesreflect basal physiologic levels of p16.

[0252] Primary prostatic adenocarcinomas revealed two distinct p16phenotypes. Most tumors were found to have undetectable or very lowlevels of p16 protein expression (Group A—57% of cases). This wasassociated with low levels or absence of INK4A exon 1α transcripts.Another group of tumors showed elevated p16 protein expression (GroupB—43%) which was consistently associated with increased INK4A exon 1αtranscripts. These findings suggest an upregulation of the INK4A-α gene,resulting in p16 protein overexpression. Patients in Group B had a moreaggressive course, demonstrated by high levels of pre-treatment PSA(P=0.018) and a sooner time to biochemical (PSA) relapse (P=0.002). Aworse prognosis for Group B is also revealed by the trending associationof p16 overexpression with higher pathologic stage.

[0253] The negative phenotype observed in Group A might correspond tothe normal physiologic state, reflecting low-to-undetectable p16 levels.Alternatively, it could be related to mutations affecting the INK4Agene, especially homozygous deletions, or methylation of the INK4A exon1α promoter region. Nevertheless, it has been reported that these eventsare infrequent in prostate cancer (17-23). Furthermore, it appears thetumors with INK4A mutations have a more aggressive clinical course(31-33). Contrary to this, in the present study, we observed that GroupA patients had a less aggressive behavior than Group B patients. Forthese reasons, we hypothesize that the negative phenotype observed inGroup A is more likely a reflection of the normal physiologic state.

[0254] The up-regulation of the INK4A-α gene, resulting in theoverexpression of p16 protein, may develop through different mechanisms.An association between increased p16 transcript and protein levels occurin tumor cell lines and certain primary neoplasms that lack functionalpRb (1, 34-37). Moreover, p16-mediated inhibition of cell cycleprogression appears to be dependent upon functional pRb (38, 39). Thesedata support an association between p16 and pRb, where absence offunctional pRb limits p16 activity and possibly promotes INK4A-αupregulation. Alternatively, enhanced activation of the INK4A-α gene mayoccur. E2F1, a direct activator of the INK4A exon 1β promoter, does notappear to directly activate INK4A-α transcription (40). However,evidence does exist for an indirect effect, as E2F1 overexpression hasbeen reported to markedly increase p16 transcripts and p16-related CKIactivity (41). Overexpression of cyclin D1 and/or of Cdk4 may alsoinfluence p16 expression, through a compensatory feedback loop wherederegulation of cyclin D/Cdk4 complexes results in increased levels ofp16 protein (28, 42). In summary, it appears that an altered RB axiscould trigger p16 overexpression in certain systems.

[0255] Cellular stress produced by replicative senescence (43-45),hyperthermia (46), and UV irradiation (47) has been reported to triggerp16 overexpression. In the present study, another type of cellularstress, androgen ablation, may account in part for this observedphenomenon. A subset of patients were treated with neoadjuvant androgenablation, a strategy reported to decrease the incidence of positivesurgical margins after prostatectomy (48). In the present study, p16overexpression was observed in 71% of hormone-treated versus 26% ofhormone-naive patients (P=0.001). These data suggest that p16 expressionmay be enhanced by androgen depletion. Androgens are known to modulatethe expression of other CKI's such as p27 and p21 (49). In addition, ithas been reported that the presence of androgens triggers downregulationof p16 in LNCaP cells (50), a finding consistent with our observation ofp16 overexpression in cases of androgen ablation. It is also possiblethat the association between p16 expression and androgen ablation may,in part, reflect staging bias by clinicians. In this setting, patientsthought to have advanced disease may have been treated with neoadjuvanttherapy.

[0256] Based on the the above referred data, it is our workinghypothesis that p16 overexpression in prostate cancer represents analtered phenotype, which identifies a subgroup of patients with a higherlikelihood of post surgical failure and tumor recurrence. In support ofthis postulate, a preliminary report in prostate cancer has demonstratedan association between p16 overexpression and poor outcome, as relatedto biochemical failure (51). In addition, p16 overexpression has beenassociated with tumor progression and a poor prognosis in ovarian (52)and breast cancers (37). Though p16 acts as a negative cell cycleregulator, specific mechanisms may contribute to its altered expression,overcoming p16-mediated tumor suppressor activities. Ongoing studies mayelucidate mechanisms of p16 overexpression relative to androgendepletion and/or alterations in the RB axis.

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What is claimed is:
 1. A method for determining the aggressiveness of aprostate carcinoma comprising: (a) obtaining a sample of the prostatecarcinoma; and (b) detecting the presence of p27 protein in the prostatecarcinoma, the absence of p27 indicating that the prostate carcinoma isaggressive.
 2. A method for diagnosing a beign prostate hyperplasiacomprising: (a) obtaining an appropriate sample of the hyperplasia; and(b) detecting the presence of the p27 RNA, a decrease of the p27 RNAindicating that the hyperplasia is beign.
 3. A method of claim 2,further comprising detecting the protein expression of p27 wherein thisadditional step may be performed before or after the detection of thepresence of the p27 RNA.
 4. A method for predicting the life-span ofpatient with prostate carcinoma comprising: (a) obtaining a sample ofthe prostate carcinoma; and (b) detecting the presence of p27 protein inthe prostate carcinoma, the presence of the p27 protein indicating thatthe patient can live longer than the patient who are undetectable p27protein.
 5. A method for increasing the life-span of patient withprostate carcinoma comprising inducing the expression of p27 protein inthe prostate carcinoma.
 6. A method for prolong life-span of patientwith prostate carcinoma which comprises introducing a nucleic acidmolecule having sequence encoding a p27 protein into the carcinoma cellunder conditions permitting expression of said gene so as to prolong thelife-span of the patient with said prostate carcinoma.
 7. The method ofclaim 6, wherein the nucleic acid molecule comprises a vector.
 8. Themethod of claim 7, wherein the vector is an adenovirus vector,adenoassociated virus vector, Epstein-Barr virus vector, retrovirusvector or vaccinia virus vector.
 9. A method for prolong life-span ofpatient with prostate carcinoma which comprises introducing an effectiveamount of p27 protein into the carcinoma cell so as to thereby prolongthe life-span of the patient with said prostate carcinoma.
 10. A methodfor prolong life-span of patient with prostate carcinoma which comprisesintroducing an effective amount of a substance capable of stabilizingthe p27 protein into the carcinoma cell so as to thereby prolong thelife-span of the patient with said prostate carcinoma.
 11. A compositionfor prolong life-span of patient with prostate carcinoma which comprisesan effective amount of a nucleic acid molecule having sequence encodinga p27 protein and a suitable carrier.
 12. A composition for prolonglife-span of patient with prostate carcinoma which comprises aneffective amount of the p27 protein and a suitable carrier.
 13. Acomposition for prolong life-span of patient with prostate carcinomawhich comprises an effective amount a substance capable of stabilizingthe p27 protein and a suitable carrier.
 14. A method for determining therate of proliferation of a prostate cancer comprising: (a) obtaining asample of the prostate cancer; and (b) detecting the presence of p21protein in the prostate cancer, the presence of p21 indicating that theprostate cancer will have a high proliferation rate.
 15. A method fordetermining the rate of proliferation of a prostate cancer comprising:(a) obtaining a sample of the prostate cancer; and (b) detecting themdm2 expression in the prostate cancer, the overexpression of mdm2indicating that the prostate cancer will have high proliferation rate.16. A method for determining whether a prostate cancer would bemetastatic comprising: (a) obtaining a sample of the prostate cancer;and (b) detecting the level of cyclin D1 expression in the prostatecancer, the overexpression of cyclin D1 indicating that the prostatecancer will be metastatic.
 17. The method of claim 16, wherein theprostate cancer is metastatic to bone.
 18. A method for determining thetumor recurrence in prostate cancer comprising: (a) obtaining a sampleof the prostate cancer; and (b) detecting the expression of thecyclin-dependent kinase inhibitor p16 in the prostate cancer, theoverexpression of p16 indicating that the prostate cancer will have hightumor recurrence.